Keratinocyte growth factor-2 polynucleotides

ABSTRACT

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a Keratinocyte Growth Factor, sometimes hereinafter referred to as “KGF-2” also formerly known as Fibroblast Growth Factor 12 (FGF-12). This invention further relates to the therapeutic use of KGF-2 to promote or accelerate wound healing. This invention also relates to novel mutant forms of KGF-2 that show enhanced activity, increased stability, higher yield or better solubility.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/610,651, filed Jun. 30, 2000 now U.S. Pat. No. 6,693,077, whichclaims benefit under 35 U.S.C. § 119(e) of U.S. Provisional ApplicationNos. 60/205,417, filed May 19, 2000, 60/198,322, filed Apr. 19, 2000,60/171,677, filed Dec. 22, 1999, 60/163,375, filed Nov. 3, 1999,60/149,935, filed Aug. 19, 1999, 60/148,628, filed Aug. 12, 1999,60/144,024, filed Jul. 15, 1999, 60/143,648, filed Jul. 14, 1999, and60/142,343, filed Jul. 2, 1999; U.S. application Ser. No. 09/610,651 isalso a continuation-in-part of U.S. application Ser. No. 09/345,373,filed Jul. 1, 1999, now U.S. Pat. No. 6,903,072 and Ser. No. 08/696,135,filed Aug. 13, 1996, now abandoned; U.S. application Ser. No. 09/345,373is a continuation of U.S. application Ser. No. 09/023,082, filed Feb.13, 1998 (now U.S. Pat. No. 6,077,692, issued Jun. 20, 2000), which is acontinuation-in-part of U.S. application Ser. No. 08/910,875, filed Aug.13, 1997 and Ser. No. 08/862,432, filed May 23, 1997; U.S. applicationSer. No. 09/023,082 also claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Nos. 60/039,045, filed Feb. 28, 1997 and60/055,561, filed Aug. 13, 1997; U.S. application Ser. No. 08/910,875claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional ApplicationNo. 60/023,852, filed Aug. 13, 1996; U.S. application Ser. No.08/862,432 is a divisional of U.S. application Ser. No. 08/461,195,filed Jun. 5, 1995, which is a continuation-in-part of InternationalApplication No. PCT/US95/01790, filed Feb. 14, 1995; U.S. applicationSer. No. 08/696,135 is a continuation-in-part of U.S. application Ser.No. 08/461,195, filed Jun. 5, 1995, which is a continuation-in-part ofInternational Application No. PCT/US95/01790, filed Feb. 14, 1995, allof which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention is a Keratinocyte Growth Factor, sometimes hereinafterreferred to as “KGF-2” also formerly known as Fibroblast Growth Factor12 (FGF-12). This invention further relates to the therapeutic use ofKGF-2 to promote or accelerate wound healing. This invention alsorelates to novel mutant forms of KGF-2 that show enhanced activity,increased stability, higher yield or better solubility. In addition,this invention relates to a method of purifying the KGF-2 polypeptide.

2. Related Art

The fibroblast growth factor family has emerged as a large family ofgrowth factors involved in soft-tissue growth and regeneration. Itpresently includes several members that share a varying degree ofhomology at the protein level, and that, with one exception, appear tohave a similar broad mitogenic spectrum, i.e., they promote theproliferation of a variety of cells of mesodermal and neuroectodermalorigin and/or promote angiogenesis.

The pattern of expression of the different members of the family is verydifferent, ranging from extremely restricted expressions of some stagesof development, to rather ubiquitous expression in a variety of tissuesand organs. All the members appear to bind heparin and heparin sulfateproteoglycans and glycosaminoglycans and strongly concentrate in theextracellular matrix. KGF was originally identified as a member of theFGF family by sequence homology or factor purification and cloning.Keratinocyte growth factor (KGF) was isolated as a mitogen for acultured murine keratinocyte line (Rubin, J. S. et al., Proc. Natl.Acad. Sci. USA 86:802–806 (1989)). Unlike the other members of the FGFfamily, it has little activity on mesenchyme-derived cells butstimulates the growth of epithelial cells. The Keratinocyte growthfactor gene encodes a 194-amino acid polypeptide (Finch, P. W. et al.,Science 245:752–755 (1989)). The N-terminal 64 amino acids are unique,but the remainder of the protein has about 30% homology to bFGF. KGF isthe most divergent member of the FGF family. The molecule has ahydrophobic signal sequence and is efficiently secreted.Post-translational modifications include cleavage of the signal sequenceand N-linked glycosylation at one site, resulting in a protein of 28kDa. Keratinocyte growth factor is produced by fibroblast derived fromskin and fetal lung (Rubin et al. (1989)). The Keratinocyte growthfactor mRNA was found to be expressed in adult kidney, colon and ilium,but not in brain or lung (Finch, P. W. et al. Science 245:752–755(1989)). KGF displays the conserved regions within the FGF proteinfamily. KGF binds to the FGF-2 receptor with high affinity.

Impaired wound healing is a significant source of morbidity and mayresult in such complications as dehiscence, anastomotic breakdown and,non-healing wounds. In the normal individual, wound healing is achieveduncomplicated. In contrast, impaired healing is associated with severalconditions such as diabetes, infection, immunosuppression, obesity andmalnutrition (Cruse, P. J. and Foord, R., Arch. Surg. 107:206 (1973);Schrock, T. R. et al., Ann. Surg. 177:513 (1973); Poole, G. U., Jr.,Surgery 97:631 (1985); Irvin, G. L. et al., Am. Surg. 51:418 (1985)).

Wound repair is the result of complex interactions and biologicprocesses. Three phases have been described in normal wound healing:acute inflammatory phase, extracellular matrix and collagen synthesis,and remodeling (Peacock, E. E., Jr., Wound Repair, 2nd edition, W BSaunders, Philadelphia (1984)). The process involves the interaction ofkeratinocytes, fibroblasts and inflammatory cells at the wound site.

Tissue regeneration appears to be controlled by specific peptide factorswhich regulate the migration and proliferation of cells involved in therepair process (Barrett, T. B. et al., Proc. Natl. Acad. Sci. USA81:6772–6774 (1985); Collins, T. et al., Nature 316:748–750 (1985)).Thus, growth factors may be promising therapeutics in the treatment ofwounds, burns and other skin disorders (Rifkin, D. B. and Moscatelli, J.Cell. Biol. 109:1–6 (1989); Sporn, M. B. et al., J. Cell. Biol.105:1039–1045 (1987); Pierce, G. F. et al., J. Cell. Biochem. 45;319–326(1991)). The sequence of the healing process is initiated during anacute inflammatory phase with the deposition of provisional tissue. Thisis followed by re-epithelialization, collagen synthesis and deposition,fibroblast proliferation, and neovascularization, all of whichultimately define the remodeling phase (Clark, R. A. F., J. Am. Acad.Dermatol. 13:701 (1985)). These events are influenced by growth factorsand cytokines secreted by inflammatory cells or by the cells localizedat the edges of the wound (Assoian, R. K. et al., Nature (Lond.) 309:804(1984); Nemeth, G. G. et al., “Growth Factors and Their Role in Woundand Fracture Healing,” Growth Factors and Other Aspects of Wound Healingin Biological and Clinical Implications, New York (1988), pp. 1–17.

Several polypeptide growth factors have been identified as beinginvolved in wound healing, including keratinocyte growth factor (KGF)(Antioniades, H. et al., Proc. Natl. Acad. Sci. USA 88:565 (1991)),platelet derived growth factor (PDGF) (Antioniades, H. et al., Proc.Natl. Acad. Sci. USA 88:565 (1991); Staiano-Coico, L. et al., Jour. Exp.Med. 178:865–878 (1993)), basic fibroblast growth factor (bFGF) (Golden,M. A. et al., J. Clin. Invest. 87:406 (1991)), acidic fibroblast growthfactor (aFGF) (Mellin, T. N. et al., J. Invest. Dermatol. 104:850–855(1995)), epidermal growth factor (EGF) (Whitby, D. J. and Ferguson, W.J., Dev. Biol. 147:207. (1991)), transforming growth factor-α (TGF-α)(Gartner, M. H. et al., Surg. Forum 42:643 (1991); Todd, R. et al., Am.J. Pathol. 138;1307 (1991)), transforming growth factor-β (TGF-β) (Wong,D. T. W. et al., Am. J. Pathol. 143:622 (1987)), neu differentiationfactor (rNDF) (Danilenko, D. M. et al., J. Clin. Invest. 95;842–851(1995)), insulin-like growth factor I (IGF-1), and insulin-like growthfactor II (IGF-II) (Cromack, D. T. et al., J. Surg. Res. 42:622 (1987)).

It has been reported that rKGF-1 in the skin stimulates epidermalkeratinocytes, keratinocytes within hair follicles and sebaceous glands(Pierce, G. F. et al., J. Exp. Med. 179:831–840 (1994)).

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the keratinocyte growth factor(KGF-2) having the amino acid sequence as shown in FIGS. 1A–1C [SEQ IDNO:2] or the amino acid sequence encoded by the cDNA clones deposited asATCC® Deposit Number 75977 on Dec. 16, 1994. The nucleotide sequencedetermined by sequencing the deposited KGF-2 clone, which is shown inFIGS. 1A–1C [SEQ ID NO:1], contains an open reading frame encoding apolypeptide of 208 amino acid residues, including an initiation codon atpositions 1–3, with a predicted leader sequence of about 35 or 36 aminoacid residues, and a deduced molecular weight of about 23.4 kDa. Theamino acid sequence of the mature KGF-2 is shown in FIGS. 1A–1C, aminoacid residues about 36 or 37 to 208 [SEQ ID NO:2].

The polypeptide of the present invention has been putatively identifiedas a member of the FGF family, more particularly the polypeptide hasbeen putatively identified as KGF-2 as a result of amino acid sequencehomology with other members of the FGF family.

In accordance with one aspect of the present invention, there areprovided novel mature polypeptides which are KGF-2 as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof. The polypeptides of thepresent invention are of human origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding human KGF-2, includingmRNAs, DNAs, cDNAs, genomic DNA, as well as antisense analogs thereof,and biologically active and diagnostically or therapeutically usefulfragments thereof.

In accordance with another aspect of the present invention, there isprovided a process for producing such polypeptide by recombinanttechniques through the use of recombinant vectors, such as cloning andexpression plasmids useful as reagents in the recombinant production ofKGF-2 proteins, as well as recombinant prokaryotic and/or eukaryotichost cells comprising a human KGF-2 nucleic acid sequence.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptide, or polynucleotideencoding such polypeptide for therapeutic purposes, for example, tostimulate epithelial cell proliferation and basal keratinocytes for thepurpose of wound healing, and to stimulate hair follicle production andhealing of dermal wounds. KGF-2 may be clinically useful in stimulatingwound healing including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resultingfrom heat exposure or chemicals, and other abnormal wound healingconditions such as uremia, malnutrition, vitamin deficiencies andcomplications associated with systemic treatment with steroids,radiation therapy and antineoplastic drugs and antimetabolites. KGF-2can be used to promote dermal reestablishment subsequent to dermal loss.

KGF-2 can be used to increase the adherence of skin grafts to a woundbed and to stimulate re-epithelialization from the wound bed. Thefollowing are types of grafts that KGF-2 could be used to increaseadherence to a wound bed: autografts, artificial skin, allografts,autodermic grafts, autoepidermic grafts, avacular grafts, Blair-Browngrafts, bone grafts, brephoplastic grafts, cutis graft, delayed graft,dermic graft, epidermic graft, fascia graft, full thickness graft,heterologous graft, xenograft, homologous graft, hyperplastic graft,lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft,omenpal graft, patch graft, pedicle graft, penetrating graft, split skingraft, or thick split graft. KGF-2 can be used to promote skin strengthand to improve the appearance of aged skin.

It is believed that KGF-2 will also produce changes in hepatocyteproliferation, and epithelial cell proliferation in the lung, breast,pancreas, stomach, small intestine, and large intestine. KGF-2 canpromote proliferation of epithelial cells such as sebocytes, hairfollicles, hepatocytes, type II pneumocytes, mucin-producing gobletcells, and other epithelial cells and their progenitors contained withinthe skin, lung, liver, and gastrointestinal tract. KGF-2 can promoteproliferation of endothelial cells, keratinocytes, and basalkeratinocytes.

KGF-2 can also be used to reduce the side effects of gut toxicity thatresult from radiation, chemotherapy treatments or viral infections.KGF-2 may have a cytoprotective effect on the small intestine mucosa.KGF-2 may also stimulate healing of mucositis (mouth ulcers) that resultfrom chemotherapy and viral infections.

KGF-2 can further be used in full regeneration of skin in full andpartial thickness skin defects, including burns, (i.e., repopulation ofhair follicles, sweat glands, and sebaceous glands), treatment of otherskin defects such as psoriasis. KGF-2 can be used to treat epidermolysisbullosa, a defect in adherence of the epidermis to the underlying dermiswhich results in frequent, open and painful blisters by acceleratingreepithelialization of these lesions. KGF-2 can also be used to treatgastric and doudenal ulcers and help heal by scar formation of themucosal lining and regeneration of glandular mucosa and duodenal mucosallining more rapidly. Inflamamatory bowel diseases, such as Crohn'sdisease and ulcerative colitis, are diseases which result in destructionof the mucosal surface of the small or large intestine, respectively.Thus, KGF-2 could be used to promote the resurfacing of the mucosalsurface to aid more rapid healing and to prevent progression ofinflammatory bowel disease. KGF-2 treatment is expected to have asignificant effect on the production of mucus throughout thegastrointestinal tract and could be used to protect the intestinalmucosa from injurious substances that are ingested or following surgery.KGF-2 can be used to treat diseases associated with the under expressionof KGF-2.

Moreover, KGF-2 can be used to prevent and heal damage to the lungs dueto various pathological states. A growth factor such as KGF-2 whichcould stimulate proliferation and differentiation and promote the repairof alveoli and brochiolar epithelium to prevent or treat acute orchronic lung damage. For example, emphysema, which results in theprogressive loss of aveoli, and inhalation injuries, i.e., resultingfrom smoke inhalation and burns, that cause necrosis of the bronchiolarepithelium and alveoli could be effectively treated with KGF-2. Also,KGF-2 could be used to stimulate the proliferation of anddifferentiation of type II pneumocytes, which may help treat or preventdisease such as hyaline membrane diseases, such as infant respiratorydistress syndrome and bronchopulmonary displasia, in premature infants.

KGF-2 could stimulate the proliferation and differentiation ofhepatocytes and, thus, could be used to alleviate or treat liverdiseases and pathologies such as fulminant liver failure caused bycirrhosis, liver damage caused by viral hepatitis and toxic substances(i.e., acetaminophen, carbon tetrachloride and other hepatotoxins knownin the art).

In addition, KGF-2 could be used treat or prevent the onset of diabetesmellitus. In patients with newly diagnosed Types I and II diabetes,where some islet cell function remains, KGF-2 could be used to maintainthe islet function so as to alleviate, delay or prevent permanentmanifestation of the disease. Also, KGF-2 could be used as an auxiliaryin islet cell transplantation to improve or promote islet cell function.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with another aspect of the present invention, there areprovided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to human KGF-2 sequences.

In accordance with a further aspect of the present invention, there areprovided mimetic peptides of KGF-2 which can be used as therapeuticpeptides. Mimetic KGF-2 peptides are short peptides which mimic thebiological activity of the KGF-2 protein by binding to and activatingthe cognate receptors of KGF-2. Mimetic KGF-2 peptides can also bind toand inhibit the cognate receptors of KGF-2.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, to reduce scarringduring the wound healing process and to prevent and/or treat tumorproliferation, diabetic retinopathy, rheumatoid arthritis,oesteoarthritis and tumor growth. KGF-2 antagonists can also be used totreat diseases associated with the over expression of KGF-2.

In accordance with yet another aspect of the present invention, thereare provided diagnostic assays for detecting diseases or susceptibilityto diseases related to mutations in KGF-2 nucleic acid sequences orover-expression of the polypeptides encoded by such sequences.

In accordance with another aspect of the present invention, there isprovided a process for utilizing such polypeptides, or polynucleotidesencoding such polypeptides, for in vitro purposes related to scientificresearch, synthesis of DNA and manufacture of DNA vectors.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the KGF-2 polypeptide having the complete amino acid sequencein FIGS. 1A–1C [SEQ ID NO:2]; (b) a nucleotide sequence encoding themature KGF-2 polypeptide having the amino acid sequence at positions 36or 37 to 208 in FIGS. 1A–1C [SEQ ID NO:2]; (c) a nucleotide sequenceencoding the KGF-2 polypeptide having the complete amino acid sequenceencoded by the cDNA clone contained in ATCC® Deposit No. 75977; (d) anucleotide sequence encoding the mature KGF-2 polypeptide having theamino acid sequence encoded by the cDNA clone contained in ATCC® DepositNo.75977; and (e) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c) or (d) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 80% identical, and more preferably at least 85%, 90%, 91%, 92%,93%, 94%, 95%, 97%, 98% or 99% identical, to any of the nucleotidesequences in (a), (b), (c), (d) or (e), above, or a polynucleotide whichhybridizes under stringent hybridization conditions to a polynucleotidein (a), (b), (c), (d) or (e), above. This polynucleotide whichhybridizes does not hybridize under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues. An additional nucleic acid embodiment ofthe invention relates to an isolated nucleic acid molecule comprising apolynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a KGF-2 having an amino acid sequence in (a),(b), (c) or (d), above.

The invention further provides an isolated KGF-2 polypeptide havingamino acid sequence selected from the group consisting of: (a) the aminoacid sequence of the KGF-2 polypeptide having the complete 208 aminoacid sequence, including the leader sequence shown in FIGS. 1A–1C [SEQID NO:2]; (b) the amino acid sequence of the mature KGF-2 polypeptide(without the leader) having the amino acid sequence at positions 36 or37 to 208 in FIGS. 1A–1C [SEQ ID NO:2]; (c) the amino acid sequence ofthe KGF-2 polypeptide having the complete amino acid sequence, includingthe leader, encoded by the cDNA clone contained in ATCC® DepositNo.75977; and (d) the amino acid sequence of the mature KGF-2polypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC® Deposit No. 75977. The polypeptides of the presentinvention also include polypeptides having an amino acid sequence withat least 80% similarity, and more preferably at least 90%, 95%, 96%,97%, 98% or 99% similarity to those described in (a), (b), (c) or (d)above, as well as polypeptides having an amino acid sequence at least80% identical, more preferably at least 85% identical, and still morepreferably 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical tothose above.

An additional aspect of the invention relates to a peptide orpolypeptide which has the amino acid sequence of an epitope-bearingportion of a KGF-2 polypeptide having an amino acid sequence describedin (a), (b), (c) or (d), above. Peptides or polypeptides having theamino acid sequence of an epitope-bearing portion of a KGF-2 polypeptideof the invention include portions of such polypeptides with at least sixor seven, preferably at least nine, and more preferably at least about30 amino acids to about 50 amino acids, although epitope-bearingpolypeptides of any length up to and including the entire amino acidsequence of a polypeptide of the invention described above also areincluded in the invention. In another embodiment, the invention providesan isolated antibody that binds specifically to a KGF-2 polypeptidehaving an amino acid sequence described in (a), (b), (c) or (d) above.

In accordance with another aspect of the present invention, novelvariants of KGF-2 are described. These can be produced by deleting orsubstituting one or more amino acids of KGF-2. Natural mutations arecalled allelic variations. Allelic variations can be silent (no changein the encoded polypeptide) or may have altered amino acid sequence. Inorder to attempt to improve or alter the characteristics of nativeKGF-2, protein engineering may be employed. Recombinant DNA technologyknown in the art can be used to create novel polypeptides. Muteins anddeletion mutations can show, e.g., enhanced activity or increasedstability. In addition, they could be purified in higher yield and showbetter solubility at least under certain purification and storageconditions.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1A–1C illustrate the cDNA and corresponding deduced amino acidsequence of the polypeptide of the present invention. The initial 35 or36 amino acid residues represent the putative leader sequence(underlined). The standard one letter abbreviations for amino acids areused. Sequencing inaccuracies are a common problem when attempting todetermine polynucleotide sequences. Sequencing was performed using a 373Automated DNA sequencer (Applied Biosystems, Inc.). Sequencing accuracyis predicted to be greater than 97% accurate. [SEQ ID NO:1]

FIGS. 2A–2D are an illustration of a comparison of the amino acidsequence of the polypeptide of the present invention and otherfibroblast growth factors. [SEQ ID NOS:13–22]

FIGS. 3A–3D show the full length mRNA and amino acid sequence for theKGF-2 gene. [SEQ ID NOS:23 and 24]

FIGS. 4A–4E show an analysis of the KGF-2 amino acid sequence. Alpha,beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues 41–109 in FIGS. 1A–1C [SEQ ID NO:2] correspond tothe shown highly antigenic regions of the KGF-2 protein. Hydrophobicregions (Hopp-Woods Plot) fall below the median line (negative values)while hydrophilic regions (Kyte-Doolittle Plot) are found above themedian line (positive values, e.g. amino acid residues 41–109). The plotis over the entire 208 amino acid ORF.

FIG. 5 shows the evaluation of KGF-2 on wound closure in the diabeticmice. Wounds were measured immediately after wounding and every day for5 consecutive days and on day 8. Percent wound closure was calculatedusing the following formula: [Area on day 1]−[Area on day 8]/[Area onday 1]. Statistical analysis performed using an unpaired t test(mean+/−SEM, n=5).

FIG. 6 shows the evaluation of KGF-2 on wound closure in thenon-diabetic mice. Wounds were measured immediately after wounding andevery day for 5 consecutive days and on day 8. Percent wound closure wascalculated using the following formula: [Area on day 1]−[Area on day8]/[Area on day 1]. Statistical analysis performed using an unpaired ttest (mean+/−SEM, n=5).

FIG. 7 shows a time course of wound closure in diabetic mice. Woundareas were measured immediately after wounding and every day for 5consecutive days and on day 8. Values are presented as total area (sq.mm). Statistical analysis performed using an unpaired t test(mean+/−SEM, n=5).

FIG. 8 shows a time course of wound closure in non-diabetic mice. Woundareas were measured immediately after wounding and every day for 5consecutive days and on day 8. Values are presented as total area (sq.mm). Statistical analysis performed using an unpaired t test(mean+/−SEM, n=5).

FIG. 9 shows a histopathologic evaluation on KGF-2 on the diabetic mice.Scores were given by a blind observer. Statistical analysis performedusing an unpaired t test (mean+/−SEM, n=5).

FIG. 10 shows a histopathologic evaluation on KGF-2 on the non-diabeticmice. Scores were given by a blind observer. Statistical analysisperformed using an unpaired t test (mean+/−SEM, n=5).

FIG. 11 shows the effect of keratinocyte growth in the diabetic mice.Scores were given by a blind observer. Statistical analysis performedusing an unpaired t test (mean+/−SEM, n=5).

FIG. 12 shows the effect of keratinocyte growth in the non-diabeticmice. Scores were given by a blind observer based. Statistical analysisperformed using an unpaired t test (mean+/−SEM, n=5).

FIG. 13 shows the effect of skin proliferation in the diabetic mice.Scores were given by a blind observer. Statisical analysis performedusing an unpaired t test (mean+/−SEM, n=5).

FIG. 14 shows the effect of skin proliferation in the non-diabetic mice.Scores were given by a blind observer. Statistical analysis performedusing an unpaired t test (mean+/−SEM, n=5).

FIG. 15 shows the DNA sequence and the protein expressed from thepQE60-Cys37 construct [SEQ ID NOS:29 and 30]. The expressed KGF-2protein contains the sequence from Cysteine at position 37 to Serine atposition 208 with a 6×(His) tag attached to the N-terminus of theprotein.

FIG. 16 shows the effect of methyl-prednisolone on wound healing inrats. Male SD adult rats (n=5) were injected on day of wounding with 5mg of methyl prednisolone. Animals received dermal punch wounds (8 mm)and were treated daily with buffer solution or KGF-2 solution in 50 μLbuffer solution for 5 consecutive days. Wounds were measured daily ondays 1–5 and on day 8 with a calibrated Jameson caliper. Valuesrepresent measurements taken on day 8. (Mean+/−SEM)

FIG. 17 shows the effect of KGF-2 on wound closure. Male SD adult rats(n=5) received dermal punch wounds (8 mm) and 5 mg ofmethyl-prednisolone on day of wounding. Animals were treated daily witha buffer solution or KGF-2 in 50 μL of buffer solution for 5 consecutivedays commencing on the day of wounding. Measurements were made daily for5 consecutive days and on day 8. Wound closure was calculated by thefollowing formula: [Area on Day 8]−[Area on Day 1]/[Area on Day 1]. Areaon day 1 was determined to be 64 sq. mm, the area made by the dermalpunch. Statistical analysis was done using an unpaired t test.(Mean+/−SEM).

FIG. 18 shows the time course of wound healing in theglucocorticoid-impaired model of wound healing. Male SD adult rats (n=5)received dermal punch wounds (8 mm) on day 1 and were treated daily for5 consecutive days with a buffer solution or a KGF-2 solution in 50 μL.Animals received 5 mg of methyl-prednisolone on day of wounding. Woundswere measured daily for five consecutive days commencing on day ofwounding and on day 8 with a calibrated Jameson caliper. Statisticalanalysis was done using an unpaired t test. (Mean+/−SEM)

FIG. 19(A) shows the effect of KGF-2 on wound area in rat model of woundhealing without methyl-prednisolone at day 5 postwounding. Male SD rats(n=5) received dermal punch wounds (8 mm) on day 1 and were treateddaily with either a buffer solution or KGF-2 in a 50 μL solution on dayof wounding and thereafter for 5 consecutive days. Wounds were measureddaily using a calibrated Jameson caliper. Statistical analysis was doneusing an unpaired t test. (Mean+/−SEM). (B) Evaluation of PDGF-BB andKGF-2 in Male SD Rats (n=6). All rats received 8 mm dorsal wounds andmethylprednisolone (MP) (17 mg/kg) to impair wound healing. Wounds weretreated daily with buffer or various concentrations of PDGF-BB andKGF-2. Wounds were measured on Days 2, 4, 6, 8, and 10 using acalibrated Jameson caliper. Statistical analysis was performed using anunpaired t-test. (Mean+/−SE) *Compared with buffer. **PDGF-BB 1 μg vsKGF-2/E3 1 μg.

FIG. 20 shows the effect of KGF-2 on wound distance in theglucocorticoid-impaired model of wound healing. Male SD adult rats (n=5)received dermal punch wounds (8 mm) and of 17 mg/kg methyl-prednisoloneon the day of wounding. Animals were treated daily with a buffersolution or KGF-2 in 50 μL of buffer solution for 5 consecutive days andon day 8. Wound distance was measured under light microscopy with acalibrated micrometer. Statistical analysis was done using an unpaired ttest. (Mean+/−SEM)

FIG. 21(A) shows the stimulation of normal primary epidermalkeratinocyte proliferation by KGF-2. (B) shows the stimulation of normalprimary epidermal keratinocyte proliferation by KGF-2 Δ33. (C) shows thestimulation of normal primary epidermal keratinocyte proliferation byKGF-2 Δ28. Human normal primary epidermal keratinocytes were incubatedwith various concentrations of KGF-2, KGF-2 Δ33 or KGF-2 Δ28 for threedays. For all three experiments alamarBlue was then added for 16 hr andthe intensity of the red color converted from alamarBlue by the cellswas measured by the difference between O.D. 570 nm and O.D. 600 nm. Foreach of the KGF-2 proteins a positive control with complete keratinocytegrowth media (KGM), and a negative control with keratinocyte basal media(KBM) were included in the same assay plate.

FIG. 22(A) shows the stimulation of thymidine incorporation by KGF-2 andFGF7 in Baf3 cells transfected with FGFR1b and FGFR2. The effects ofKGF-2 (right panel) and FGF7 (left panel) on the proliferation of Baf3cells transfected with FGFR1iiib (open circle) or FGFR2iiib/KGFR (solidcircle) were examined. Y-axis represents the amount of [3H]thymidineincorporation (cpm) into DNA of Baf3 cells. X-axis represents the finalconcentration of KGF-2 or FGF7 added to the tissue culture media. (B)shows the stimulation of thymidine incorporation by KGF-2 Δ33 in Baf3cells transfected with FGFR2iiib (C) shows the stimulation of thymidineincorporation by KGF-2 (white bar), KGF-2 Δ33 (black bar) and KGF-2 Δ28(grey bar) in Baf3 cells transfected with FGFR2iiib.

FIG. 23 shows the DNA and protein sequence [SEQ ID NOS:38 and 39] forthe E. coli optimized full length KGF-2.

FIGS. 24A and B show the DNA and protein sequences [SEQ ID NOS:42, 43,54, and 55] for the E. coli optimized mature KGF-2.

FIG. 25 shows the DNA and the encoded protein sequence [SEQ ID NOS:65and 66] for the KGF-2 deletion construct comprising amino acids 36 to208 of KGF-2.

FIG. 26 shows the DNA and the encoded protein sequence [SEQ ID NOS:67and 68] for the KGF-2 deletion construct comprising amino acids 63 to208 of KGF-2.

FIG. 27 shows the DNA and the encoded protein sequence [SEQ ID NOS:69and 70] for the KGF-2 deletion construct comprising amino acids 77 to208 of KGF-2.

FIG. 28 shows the DNA and the encoded protein sequence [SEQ ID NOS:71and 72] for the KGF-2 deletion construct comprising amino acids 93 to208 of KGF-2.

FIG. 29 shows the DNA and the encoded protein sequence [SEQ ID NOS:73and 74] for the KGF-2 deletion construct comprising amino acids 104 to208 of KGF-2.

FIG. 30 shows the DNA and the encoded protein sequence [SEQ ID NOS:75and 76] for the KGF-2 deletion construct comprising amino acids 123 to208 of KGF-2.

FIG. 31 shows the DNA and the encoded protein sequence [SEQ ID NOS:77and 78] for the KGF-2 deletion construct comprising amino acids 138 to208 of KGF-2.

FIG. 32 shows the DNA and the encoded protein sequence [SEQ ID NOS:79and 80] for the KGF-2 deletion construct comprising amino acids 36 to153 of KGF-2.

FIG. 33 shows the DNA and the encoded protein sequence [SEQ ID NOS:81and 82] for the KGF-2 deletion construct comprising amino acids 63 to153 of KGF-2.

FIG. 34 shows the DNA sequence for the KGF-2 Cysteine-37 to Serinemutant construct [SEQ ID NO:83].

FIG. 35 shows the DNA sequence for the KGF-2 Cysteine-37/Cysteine-106 toSerine mutant construct [SEQ ID NO:84].

FIG. 36 shows the evaluation of KGF-2 Δ33 effects on wound healing inmale SD rats (n=5). Animals received 6 mm dorsal wounds and were treatedwith various concentrations of buffer, or KGF-2 Δ33 for 4 consecutivedays. Wounds were measured daily using a calibrated Jameson caliper.Statistical analysis was done using an unpaired t-test.(Mean+/−SE)

*Compared with buffer.

FIG. 37 shows the effect of KGF-2 Δ33 on wound healing in normal rats.Male, SD, 250–300 g, rats (n=5) were given 6 mm full-thickness dorsalwounds. Wounds were measured with a caliper and treated with variousconcentrations of KGF-2Δ33 and buffer for four days commencing on theday of surgery. On the final day, wounds were harvested. Statisticalanalysis was performed using an unpaired t-test. *Value is compared toNo Treatment Control. †value is compared to Buffer Control.

FIG. 38 shows the effect of KGF-2 Δ33 on breaking strength in incisionalwounds. Male adult SD rats (n=10) received 2.5 cm full thicknessincisional wounds on day 1 and were intraincisionally treatedpostwounding with one application of either buffer or KGF-2 (Delta 33)(1, 4, and 10 μg). Animals were sacrificed on day 5 and 0.5 cm woundspecimens were excised for routine histology and breaking strengthanalysis. Biomechanical testing was accomplished using an Instron skintensiometer with a force applied across the wound. Breaking strength wasdefined as the greatest force withheld by each wound prior to rupture.Statistical analysis was done using an unpaired t-test. (Mean+/−SE).

FIG. 39 shows the effect of KGF-2 (Delta 33) on epidermal thickness inincisional wounds. Male adult SD rats (n=10) received 2.5 cm fullthickness incisional wounds on day 1 and were intracisionally treatedpostwounding with one application of either buffer or KGF-2 (Delta 33)(1, 4, and 10 μg). Animals were sacrificed on day 5 and 0.5 cm woundspecimens were excised for routine histology and breaking strengthanalysis. Epidermal thickness was determined by taking the mean of 6measurements taken around the wound site. Measurements were taken by ablind observer on Masson Trichrome stained sections under lightmicroscopy using a calibrated lens micrometer. Statistical analysis wasdone using an unpaired t-test. (Mean+/−SE).

FIG. 40 shows the effect of KGF-2 (Delta 33) on epidermal thicknessafter a single intradermal injection. Male adult SD rats (n=18) received6 intradermal injections of either buffer or KGF-2 in a concentration of1 and 4 μg in 50 μL on day 0. Animals were sacrificed 24 and 48 hourspost injection. Epidermal thickness was measured from the granular layerto the bottom of the basal layer. Approximately 20 measurements weremade along the injection site and the mean thickness quantitated.Measurements were determined using a calibrated micrometer on MassonTrichrome stained sections under light microscopy. Statistical analysiswas done using an unpaired t-test. (Mean+/−SE).

FIG. 41 shows the effect of KGF-2 (Delta 33) on BrdU scoring. Male adultSD rats (n=18) received 6 intradermal injections of either placebo orKGF-2 in a concentration of 1 and 4 μg in 50 μL on day 0. Animals weresacrificed 24 and 48 hours post injection. Animals were injected with5–2′-Bromo-deoxyrudine (100 mg/kg ip) two hours prior to sacrifice.Scoring was done by a blinded observer under light microscopy using thefollowing scoring system: 0–3 none to minimal BrdU labeled cells; 4–6moderate labeling; 7–10 intense labeled cells. Statistical analysis wasdone using an unpaired t-test. (Mean+/−SE).

FIG. 42 shows the anti-inflammatory effect of KGF-2 on PAF-induced pawedema.

FIG. 43 shows the anti-inflammatory effect of KGF-2 Δ33 on PAF-inducedpaw edema in Lewis rats.

FIG. 44 shows the effect of KGF-2 Δ33 on the survival of whole bodyirradiated Balb/c mice. Balb/c male mice (n=5), 22.1 g were irradiatedwith 519 RADS. Animals were treated with buffer or KGF-2 (1 & 5 mg/kg,s.q.) 2 days prior to irradiation and daily thereafter for 7 days.

FIG. 45 shows the effect of KGF-2 Δ33 on body weight of irradiated mice.Balb/c male mice (n=5) weighing 22.1 g were injected with either Bufferor KGF-2_(—)33 (1,5 mg/kg) for 2 days prior to irradiation with 519Rad/min. The animals were weighed daily and injected for 7 daysfollowing irradiation.

FIG. 46 shows the effect of KGF-2 Δ33 on the survival rate of whole bodyirradiated Balb/c mice. Balb/c male mice (n=7), 22.1 g were irradiatedwith 519 RADS. Animals were treated with buffer or KGF-2 (1 and 5 mg/kg,s.q.) 2 days prior to irradiation and daily thereafter for 7 days.

FIG. 47 shows the effect of KGF-2 Δ33 on wound healing in aglucocorticoid-impaired rat model.

FIG. 48 shows the effect of KGF-2 Δ33 on cell proliferation asdetermined using BrdU labeling.

FIG. 49 shows the effect of KGF-2 Δ33 on the collagen content localizedat anastomotic surgical sites in the colons of rats.

FIG. 50 shows a schematic representation of the pHE4-5 expression vector(SEQ ID NO:147) and the subcloned KGF-2 cDNA coding sequence. Thelocations of the kanamycin resistance marker gene, the KGF-2 codingsequence, the oriC sequence, and the lacIq coding sequence areindicated.

FIG. 51 shows the nucleotide sequence of the regulatory elements of thepHE promoter (SEQ ID NO:148). The two lac operator sequences, theShine-Delgarno sequence (S/D), and the terminal HindIII and NdeIrestriction sites (italicized) are indicated.

FIG. 52 shows the proliferation of bladder epithelium following ip or scadministration of KGF-2 Δ33.

FIG. 53 shows the proliferation of prostatic epithelial cells aftersystemic administration of KGF-2 Δ33.

FIG. 54 shows the effect of KGF-2 Δ33 on bladder wall ulceration in acyclophosphamide-induced hemorrhagic cystitis model in the rat.

FIG. 55 shows the effect of KGF-2 Δ33 on bladder wall thickness in acyclophosphamide-induced cystitis rat model.

FIG. 56 provides an overview of the study design to determine whetherKGF-2 Δ33 induces proliferation of normal epithelia in rats whenadministered systemically using SC and IP routes.

FIG. 57. Normal Sprague Dawley rats were injected daily with KGF-2 Δ33(5 mg/kg; HG03411-E2) or buffer and sacrificed one day after the finalinjection. A blinded observer counted the proliferating cells in tenrandomly chosen fields per animals at a 10× magnification. SCadministration of KGF-2 Δ33 elicited a significant proliferation afterone day which then returned to normal by 2 days. KGF-2 Δ33 given ipstimulated proliferation from 1–3 days but only the results from days 1and 3 were statistically significant.

FIG. 58. Normal Sprague Dawley rats were injected daily with KGF-2 Δ33(5 mg/kg; HG0341 1-E2) or buffer and sacrificed one day after the finalinjection. A blinded observer counted the proliferating cells in tenrandomly chosen fields per animal at a 10× magnification. KGF-2 Δ33given ip stimulated proliferation over the entire study period while scadministration of KGF-2 Δ33 did not increase the proliferation at anytime point.

FIG. 59. Normal Sprague Dawley rats were injected daily with KGF-2 Δ33(5 mg/kg; HG03411-E2) or buffer and sacrificed one day after the finalinjection. A blinded observer counted the proliferating cells in onecross-section per animal at a 10× magnification. KGF-2 Δ33 given scelicited a significant increase in proliferation after 1, 2, and 3 daysof daily administration. When KGF-2 Δ33 was given ip, proliferation wasseen after 2 and 3 days only.

FIG. 60 demonstrates KGF-2 Δ33 induced proliferation in normal rat lung.

DETAILED DESCRIPTION

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for thepolypeptide having the deduced amino acid sequence of FIGS. 1A–1C (SEQID NO:2) or for the polypeptide encoded by the cDNA of the clonedeposited as ATCC® Deposit No. 75977 on Dec. 16, 1994 at the AmericanType Culture Collection Patent Depository, 10801 University Boulevard,Manassas, Va. 20110-2209 or the polypeptide encoded by the cDNA of theclone deposited as ATCC® Deposit No. 75901 on Sep. 29, 1994 at theAmerican Type Culture Collection Patent Depository, 10801 UniversityBoulevard, Manassas, Va. 20110-2209.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Unless otherwise indicated, each “nucleotide sequence” set forth hereinis presented as a sequence of deoxyribonucleotides (abbreviated A, G, Cand T). However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U), where each thymidine deoxyribonucleotide (T) in the specifieddeoxyribonucleotide sequence is replaced by the ribonucleotide uridine(U). For instance, reference to an RNA molecule having the sequence ofSEQ ID NO:1 set forth using deoxyribonucleotide abbreviations isintended to indicate an RNA molecule having a sequence in which eachdeoxyribonucleotide A, G or C of SEQ ID NO:1 has been replaced by thecorresponding ribonucleotide A, G or C, and each deoxyribonucleotide Thas been replaced by a ribonucleotide U.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) with an initiationcodon at positions 1–3 of the nucleotide sequence shown in FIGS. 1A–1C(SEQ ID NO:1); DNA molecules comprising the coding sequence for themature KGF-2 protein shown in FIGS. 1A–1C (last 172 or 173 amino acids)(SEQ ID NO:2); and DNA molecules which comprise a sequence substantiallydifferent from those described above but which, due to the degeneracy ofthe genetic code, still encode the KGF-2 protein. Of course, the geneticcode is well known in the art. Thus, it would be routine for one skilledin the art to generate the degenerate variants described above.

A polynucleotide encoding a polypeptide of the present invention may beobtained from a human prostate and fetal lung. A fragment of the cDNAencoding the polypeptide was initially isolated from a library derivedfrom a human normal prostate. The open reading frame encoding the fulllength protein was subsequently isolated from a randomly primed humanfetal lung cDNA library. It is structurally related to the FGF family.It contains an open reading frame encoding a protein of 208 amino acidresidues of which approximately the first 35 or 36 amino acid residuesare the putative leader sequence such that the mature protein comprises173 or 172 amino acids. The protein exhibits the highest degree ofhomology to human keratinocyte growth factor with 45% identity and 82%similarity over a 206 amino acid stretch. It is also important thatsequences that are conserved through the FGF family are found to beconserved in the protein of the present invention.

In addition, results from nested PCR of KGF-2 cDNA from libraries showedthat there were potential alternative spliced forms of KGF-2.Specifically, using primers flanking the N-terminus of the open readingframe of KGF-2, PCR products of 0.2 kb and 0.4 kb were obtained fromvarious cDNA libraries. A 0.2 kb size was the expected product for KGF-2while the 0.4 kb size may result from an alternatively spliced form ofKGF-2. The 0.4 kb product was observed in libraries from stomach cancer,adult testis, duodenum and pancreas.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIGS. 1A–1C (SEQ ID NO:1) or that of thedeposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same mature polypeptide as the DNA of FIGS. 1A–1C (SEQID NO:1) or the deposited cDNA.

The polynucleotide which encodes for the predicted mature polypeptide ofFIGS. 1A–1C (SEQ ID NO:2) or for the predicted mature polypeptideencoded by the deposited cDNA may include: only the coding sequence forthe mature polypeptide; the coding sequence for the mature polypeptideand additional coding sequence such as a leader or secretory sequence ora proprotein sequence; the coding sequence for the mature polypeptide(and optionally additional coding sequence) and non-coding sequence,such as intron or non-coding sequence 5′ and/or 3′ of the codingsequence for the predicted mature polypeptide. In addition, a fulllength mRNA has been obtained which contains 5′ and 3′ untranslatedregions of the gene (FIG. 3 (SEQ ID NO:23)).

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, as well as the variability ofcleavage sites for leaders in different known proteins, the actual KGF-2polypeptide encoded by the deposited cDNA comprises about 208 aminoacids, but may be anywhere in the range of 200–220 amino acids; and theactual leader sequence of this protein is about 35 or 36 amino acids,but may be anywhere in the range of about 30 to about 40 amino acids.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIGS. 1A–1C (SEQ ID NO. 2) or the polypeptide encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a nonnaturallyoccurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samepredicted mature polypeptide as shown in FIGS. 1A–1C (SEQ ID NO:2) orthe same predicted mature polypeptide encoded by the cDNA of thedeposited clone as well as variants of such polynucleotides whichvariants encode for a fragment, derivative or analog of the polypeptideof FIGS. 1A–1C (SEQ ID NO:2) or the polypeptide encoded by the cDNA ofthe deposited clone. Such nucleotide variants include deletion variants,substitution variants and addition or insertion variants.

The present invention includes polynucleotides encoding mimetic peptidesof KGF-2 which can be used as therapeutic peptides. Mimetic KGF-2peptides are short peptides which mimic the biological activity of theKGF-2 protein by binding to and activating the cognate receptors ofKGF-2. Mimetic KGF-2 peptides can also bind to and inhibit the cognatereceptors of KGF-2. KGF-2 receptors include, but are not limited to,FGFR2iiib and FGFR1iiib. Such mimetic peptides are obtained from methodssuch as, but not limited to, phage display or combinatorial chemistry.For example the method disclosed by Wrighton et al., Science 273:458–463(1996) to generate mimetic KGF-2 peptides.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIGS. 1A–1C (SEQ ID NO:1) or of the coding sequence of thedeposited clone. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encode polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both prosequence and a presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexahistidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I. et al. Cell 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or cDNA to determine whichmembers of the library the probe hybridizes to.

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 80% identical, and more preferably at least 85%, 90%, 91%, 92%,93%, 94%, 95%, 97%, 98% or 99% identical to (a) a nucleotide sequenceencoding the full-length KGF-2 polypeptide having the complete aminoacid sequence in FIGS. 1A–1C (SEQ ID NO:2), including the predictedleader sequence; (b) a nucleotide sequence encoding the mature KGF-2polypeptide (full-length polypeptide with the leader removed) having theamino acid sequence at positions about 36 or 37 to 208 in FIGS. 1A–1C(SEQ ID NO:2); (c) a nucleotide sequence encoding the full-length KGF-2polypeptide having the complete amino acid sequence including the leaderencoded by the cDNA clone contained in ATCC® Deposit No. 75977; (d) anucleotide sequence encoding the mature KGF-2 polypeptide having theamino acid sequence encoded by the cDNA clone contained in ATCC® DepositNo. 75977; (e) a nucleotide sequence encoding any of the KGF-2 analogsor deletion mutants described below; or (f) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),or (e).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a KGF-2polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the KGF-2polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99%identical to, for instance, the nucleotide sequence shown in FIGS. 1A–1C(SEQ ID NO:1) or to the nucleotides sequence of the deposited cDNA clonecan be determined conventionally using known computer programs such asthe Bestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). Bestfit uses the local homology algorithmof Smith and Waterman, Advances in Applied Mathematics 2: 482–489(1981), to find the best segment of homology between two sequences. Whenusing Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence and that gapsin homology of up to 5% of the total number of nucleotides in thereference sequence are allowed.

A preferred method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (Comp. App. Biosci. (1990) 6:237–245.) In a sequencealignment the query and subject sequences are both DNA sequences. An RNAsequence can be compared by converting U's to T's. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB alignment of DNA sequences to calculate percentidentity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, JoiningPenalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5,Gap Size Penalty=0.05, Window Size=500 or the length of the subjectnucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The present application is directed to nucleic acid molecules at least80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to thenucleic acid sequence shown in FIGS. 1A–1C [SEQ ID NO:1] or to thenucleic acid sequence of the deposited cDNA, irrespective of whetherthey encode a polypeptide having KGF-2 activity. This is because evenwhere a particular nucleic acid molecule does not encode a polypeptidehaving KGF-2 activity, one of skill in the art would still know how touse the nucleic acid molecule, for instance, as a hybridization probe ora polymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving KGF-2 activity include, inter alia, (1) isolating the KGF-2 geneor allelic variants thereof in a cDNA library; (2) in situ hybridization(e.g., “FISH”) to metaphase chromosomal spreads to provide precisechromosomal location of the KGF-2 gene, as described in Verma et al.,Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, NewYork (1988); and Northern Blot analysis for detecting KGF-2 mRNAexpression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to thenucleic acid sequence shown in FIGS. 1A–1C [SEQ ID NO:1] or to thenucleic acid sequence of the deposited cDNA which do, in fact, encode apolypeptide having KGF-2 protein activity. By “a polypeptide havingKGF-2 activity” is intended polypeptides exhibiting activity similar,but not necessarily identical, to an activity of the wild-type KGF-2protein of the invention or an activity that is enhanced over that ofthe wild-type KGF-2 protein (either the full-length protein or,preferably, the mature protein), as measured in a particular biologicalassay.

Assays of KGF-2 activity are disclosed, for example, in Examples 10 and11 below. These assays can be used to measure KGF-2 activity ofpartially purified or purified native or recombinant protein.

KGF-2 stimulates the proliferation of epidermal keratinocyes but notmesenchymal cells such as fibroblasts. Thus, “a polypeptide having KGF-2protein activity” includes polypeptides that exhibit the KGF-2 activity,in the keratinocyte proliferation assay set forth in Example 10 and willbind to the FGF receptor isoforms 1-iiib and 2-iiib (Example 11).Although the degree of activity need not be identical to that of theKGF-2 protein, preferably, “a polypeptide having KGF-2 protein activity”will exhibit substantially similar activity as compared to the KGF-2protein (i.e., the candidate polypeptide will exhibit greater activityor not more than about tenfold less and, preferably, not more than abouttwofold less activity relative to the reference KGF-2 protein).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 97%, 98% or 99% identical to the nucleic acidsequence of the deposited cDNA or the nucleic acid sequence shown inFIGS. 1A–1C [SEQ ID NO:1] will encode a polypeptide “having KGF-2protein activity.” In fact, since degenerate variants of thesenucleotide sequences all encode the same polypeptide, this will be clearto the skilled artisan even without performing the above describedcomparison assay. It will be further recognized in the art that, forsuch nucleic acid molecules that are not degenerate variants, areasonable number will also encode a polypeptide having KGF-2 proteinactivity. This is because the skilled artisan is fully aware of aminoacid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306–1310 (1990), wherein the authors indicate that thereare two main approaches for studying the tolerance of an amino acidsequence to change. The first method relies on the process of evolution,in which mutations are either accepted or rejected by natural selection.The second approach uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene and selections or screensto identify sequences that maintain functionality. As the authors state,these studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions. The authors further indicate which amino acidchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require nonpolarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie, J. U. et al., supra, and the references cited therein.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 80%, and more preferably at least 85% and still morepreferably 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIGS. 1A–1C (SEQ ID NO:1)or the deposited cDNA(s).

An example of “stringent hybridization conditions” includes overnightincubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65° C. Alternatively, the polynucleotide may have at least 20bases, preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO:1, for example, for recoveryof the polynucleotide or as a diagnostic probe or as a PCR primer.

Also contemplated are nucleic acid molecules that hybridize to the KGF-2polynucleotides at moderately high stringency hybridization conditions.Changes in the stringency of hybridization and signal detection areprimarily accomplished through the manipulation of formamideconcentration (lower percentages of formamide result in loweredstringency); salt conditions, or temperature. For example, moderatelyhigh stringency conditions include an overnight incubation at 3° C. in asolution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA;followed by washes at 50° C. with 1×SSPE, 0.1% SDS. In addition, toachieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited cDNA clone), for instance,a portion 50–750 nt in length, or even to the entire length of thereference polynucleotide, are also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the nucleotide sequence of the deposited cDNA or the nucleotidesequence as shown in FIGS. 1A–1C [SEQ ID NO:1]. By a portion of apolynucleotide of “at least 20 nt in length,” for example, is intended20 or more contiguous nucleotides from the nucleotide sequence of thereference polynucleotide (e.g., the deposited cDNA or the nucleotidesequence as shown in FIGS. 1A–1C [SEQ ID NO:1]). As indicated, suchportions are useful diagnostically either as a probe according toconventional DNA hybridization techniques or as primers foramplification of a target sequence by the polymerase chain reaction(PCR), as described, for instance, in Molecular Cloning, A LaboratoryManual, 2nd. edition, edited by Sambrook, J., Fritsch, E. F. andManiatis, T., (1989), Cold Spring Harbor Laboratory Press, the entiredisclosure of which is hereby incorporated herein by reference.

Since a KGF-2 cDNA clone has been deposited and its determinednucleotide sequence is provided in FIGS. 1A–1C [SEQ ID NO:1], generatingpolynucleotides which hybridize to a portion of the KGF-2 cDNA moleculewould be routine to the skilled artisan. For example, restrictionendonuclease cleavage or shearing by sonication of the KGF-2 cDNA clonecould easily be used to generate DNA portions of various sizes which arepolynucleotides that hybridize to a portion of the KGF-2 cDNA molecule.Alternatively, the hybridizing polynucleotides of the present inventioncould be generated synthetically according to known techniques. Ofcourse, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of the KGF-2 cDNA shown in FIGS.1A–1C [SEQ ID NO:1]), or to a complementary stretch of T (or U) resides,would not be included in a polynucleotide of the invention used tohybridize to a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

The invention further provides isolated nucleic acid moleculescomprising a polynucleotide encoding an epitope-bearing portion of theKGF-2 protein. In particular, isolated nucleic acid molecules areprovided encoding polypeptides comprising the following amino acidresidues in FIGS. 1A–1C (SEQ ID NO:2), which the present inventors havedetermined are antigenic regions of the KGF-2 protein:

1. Gly41-Asn71: GQDMVSPEATNSSSSSFSSPSSAGRHVRSYN; [SEQ ID NO:25] 2.Lys91-Ser109: KIEKNGKVSGTKKENCPYS; [SEQ ID NO:26] 3. Asn135-Tyr164:NKKGKLYGSKEFNNDCKLKERIEENGYNTY; [SEQ ID NO 27] and 4. Asn181-Ala199:NGKGAPRRGQKTRRKNTSA. [SEQ ID NO:28]

Also, there are two additional shorter predicted antigenic areas,Gln74-Arg78 of FIGS. 1A–1C (SEQ ID NO:2) and Gln170-Gln175 of FIG. 1(SEQ ID NO:2). Methods for generating such epitope-bearing portions ofKGF-2 are described in detail below.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. § 112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

KGF-2 Polypeptides and Fragments

The present invention further relates to a polypeptide which has thededuced amino acid sequence of FIGS. 1A–1C (SEQ ID NO:2) or which hasthe amino acid sequence encoded by the deposited cDNA, as well asfragments, analogs and derivatives of such polypeptide.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, as well as the variability ofcleavage sites for leaders in different known proteins, the actual KGF-2polypeptide encoded by the deposited cDNA comprises about 208 aminoacids, but may be anywhere in the range of 200–220 amino acids; and theactual leader sequence of this protein is about 35 or 36 amino acids,but may be anywhere in the range of about 30 to about 40 amino acids.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide, of FIGS. 1A–1C (SEQ ID NO:2) or that encoded by thedeposited cDNA, means a polypeptide which retains essentially the samebiological function or activity as such polypeptide. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIGS. 1A–1C(SEQ ID NO:2) or that encoded by the deposited cDNA may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

The terms “peptide” and “oligopeptide” are considered synonymous (as iscommonly recognized) and each term can be used interchangeably as thecontext requires to indicate a chain of at least two amino acids coupledby peptidyl linkages. The word “polypeptide” is used herein for chainscontaining more than ten amino acid residues. All oligopeptide andpolypeptide formulas or sequences herein are written from left to rightand in the direction from amino terminus to carboxy terminus.

It will be recognized in the art that some amino acid sequences of theKGF-2 polypeptide can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity. In general, it ispossible to replace residues which form the tertiary structure, providedthat residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

Thus, the invention further includes variations of the KGF-2 polypeptidewhich show substantial KGF-2 polypeptide activity or which includeregions of KGF-2 protein such as the protein portions discussed below.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions (for example, substituting one hydrophilic residuefor another, but not strongly hydrophilic for strongly hydrophobic as arule). Small changes or such “neutral” amino acid substitutions willgenerally have little effect on activity.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Ile;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe and Tyr.

As indicated in detail above, further guidance concerning which aminoacid changes are likely to be phenotypically silent (i.e., are notlikely to have a significant deleterious effect on a function) can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306–1310(1990).

The present invention includes mimetic peptides of KGF-2 which can beused as therapeutic peptides. Mimetic KGF-2 peptides are short peptideswhich mimic the biological activity of the KGF-2 protein by binding toand activating the cognate receptors of KGF-2. Mimetic KGF-2 peptidescan also bind to and inhibit the cognate receptors of KGF-2. KGF-2receptors include, but are not limited to, FGFR2iiib and FGFR1iiib. Suchmimetic peptides are obtained from methods such as, but not limited to,phage display or combinatorial chemistry. For example, the methoddisclosed by Wrighton et al. Science 273:458–463 (1996) can be used togenerate mimetic KGF-2 peptides.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention are preferably in an isolatedform. By “isolated polypeptide” is intended a polypeptide removed fromits native environment. Thus, a polypeptide produced and/or containedwithin a recombinant host cell is considered isolated for purposes ofthe present invention. Also intended are polypeptides that have beenpurified, partially or substantially, from a recombinant host cell or anative source.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or99% similarity (more preferably at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 97%, 98% or 99% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide (such as the deletion mutants described below) generallycontaining at least 30 amino acids and more preferably at least 50 aminoacids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2: 482–489, 1981) tofind the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a KGF-2polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the KGF-2 polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical to, forinstance, the amino acid sequence shown in FIGS. 1A–1C [SEQ ID NO:2] orto the amino acid sequence encoded by deposited cDNA clone can bedetermined conventionally using known computer programs such the Bestfitprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, 575 Science Drive,Madison, Wis. 53711). When using Bestfit or any other sequence alignmentprogram to determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

A preferred method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (Comp. App. Biosci. (1990) 6:237–245). In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

As described in detail below, the polypeptides of the present inventioncan be used to raise polyclonal and monoclonal antibodies, which areuseful in diagnostic assays for detecting KGF-2 protein expression asdescribed below or as agonists and antagonists capable of enhancing orinhibiting KGF-2 protein function. Further, such polypeptides can beused in the yeast two-hybrid system to “capture” KGF-2 protein bindingproteins which are also candidate agonist and antagonist according tothe present invention. The yeast two hybrid system is described inFields and Song, Nature 340:245–246 (1989).

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. These immunogenic epitopes arebelieved to be confined to a few loci on the molecule. On the otherhand, a region of a protein molecule to which an antibody can bind isdefined as an “antigenic epitope.” The number of immunogenic epitopes ofa protein generally is less than the number of antigenic epitopes. See,for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998–4002(1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) Antibodies that react withpredetermined sites on proteins. Science 219:660–666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals. Peptides that are extremely hydrophobic and those ofsix or fewer residues generally are ineffective at inducing antibodiesthat bind to the mimicked protein; longer, soluble peptides, especiallythose containing proline residues, usually are effective. Sutcliffe etal., supra, at 661. For instance, 18 of 20 peptides designed accordingto these guidelines, containing 8–39 residues covering 75% of thesequence of the influenza virus hemagglutinin HA1 polypeptide chain,induced antibodies that reacted with the HA1 protein or intact virus;and 12/12 peptides from the MuLV polymerase and 18/18 from the rabiesglycoprotein induced antibodies that precipitated the respectiveproteins.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. Sutcliffe et al., supra, at663. The antibodies raised by antigenic epitope-bearing peptides orpolypeptides are useful to detect the mimicked protein, and antibodiesto different peptides may be used for tracking the fate of variousregions of a protein precursor which undergoes post-translationalprocessing. The peptides and anti-peptide antibodies may be used in avariety of qualitative or quantitative assays for the mimicked protein,for instance in competition assays since it has been shown that evenshort peptides (e.g., about 9 amino acids) can bind and displace thelarger peptides in immunoprecipitation assays. See, for instance, Wilsonet al., Cell 37:767–778 (1984) at 777. The anti-peptide antibodies ofthe invention also are useful for purification of the mimicked protein,for instance, by adsorption chromatography using methods well known inthe art.

Antigenic epitope-bearing peptides and polypeptides of the inventiondesigned according to the above guidelines preferably contain a sequenceof at least seven, more preferably at least nine and most preferablybetween about 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention. However, peptides orpolypeptides comprising a larger portion of an amino acid sequence of apolypeptide of the invention, containing about 30, 40, 50, 60, 70, 80,90, 100, or 150 amino acids, or any length up to and including theentire amino acid sequence of a polypeptide of the invention, also areconsidered epitope-bearing peptides or polypeptides of the invention andalso are useful for inducing antibodies that react with the mimickedprotein. Preferably, the amino acid sequence of the epitope-bearingpeptide is selected to provide substantial solubility in aqueoussolvents (i.e., the sequence includes relatively hydrophilic residuesand highly hydrophobic sequences are preferably avoided); and sequencescontaining proline residues are particularly preferred.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate KGF-2-specific antibodies include the following:

1. Gly41-Asn71: GQDMVSPEATNSSSSSFSSPSSAGRHVRSYN; [SEQ ID NO: 25] 2.Lys91-Ser109: KIEKNGKVSGTKKENCPYS; [SEQ ID NO: 26] 3. Asn135-Tyr164:NKKGKLYGSKEFNNDCKLKERIEENGYNTY; [SEQ ID NO: 27] and 4. Asn181-Ala199:NGKGAPRRGQKTRRKNTSA. [SEQ ID NO: 28]Also, there are two additional shorter predicted antigenic areas,Gln74-Arg78 of FIGS. 1A–1C (SEQ ID NO:2) and Gln170-Gln175 of FIGS.1A–1C (SEQ ID NO:2).

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using nucleic acid molecules of theinvention. For instance, a short epitope-bearing amino acid sequence maybe fused to a larger polypeptide which acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies. Epitope-bearing peptides also may besynthesized using known methods of chemical synthesis. For instance,Houghten has described a simple method for synthesis of large numbers ofpeptides, such as 10–20 mg of 248 different 13 residue peptidesrepresenting single amino acid variants of a segment of the HA1polypeptide which were prepared and characterized (by ELISA-type bindingstudies) in less than four weeks. Houghten, R. A. (1985) General methodfor the rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids. Proc. Natl. Acad. Sci. USA 82:5131–5135. This “SimultaneousMultiple Peptide Synthesis (SMPS)” process is further described in U.S.Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure theindividual resins for the solid-phase synthesis of various peptides arecontained in separate solvent-permeable packets, enabling the optimaluse of the many identical repetitive steps involved in solid-phasemethods. A completely manual procedure allows 500–1000 or more synthesesto be conducted simultaneously. Houghten et al., supra, at 5134.

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC® DepositNo. 75977 or encoded by a polynucleotide that hybridizes to thecomplement of the sequence of SEQ ID NO:1 or contained in ATCC® DepositNo. 75977 under stringent hybridization conditions or lower stringencyhybridization conditions as defined supra. The present invention furtherencompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of the invention (such as, for example, thesequence disclosed in SEQ ID NO:1) polynucleotide sequences of thecomplementary strand of a polynucleotide sequence encoding an epitope ofthe invention, and polynucleotide sequences which hybridize to thecomplementary strand under stringent hybridization conditions or lowerstringency hybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998–4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131–5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additionally preferred antigenic epitopes comprise,or alternatively consist of, the amino acid sequence of residues: M-1 toH-15; W-2 to L-16; K-3 to P-17; W-4 to G-18; 1-5 to C-19; L-6 to C-20;T-7 to C-21; H-8 to C-22; C-9 to C-23; A-10 to F-24; S-11 to L-25; A-12to L-26; F-13 to L-27; P-14 to F-28; H-15 to L-29; L-16 to V-30; P-17 toS-31; G-18 to S-32; C-19 to V-33; C-20 to P-34; C-21 to V-35; C-22 toT-36; C-23 to C-37; F-24 to Q-38; L-25 to A-39; L-26 to L-40; L-27 toG-41; F-28 to Q-42; L-29 to D-43; V-30 to M-44; S-31 to V-45; S-32 toS-46; V-33 to P-47; P-34 to E-48; V-35 to A-49; T-36 to T-50; C-37 toN-51; Q-38 to S-52; A-39 to S-53; L-40 to S-54; G-41 to S-55; Q-42 toS-56; D-43 to F-57; M-44 to S-58; V-45 to S-59; S-46 to P-60; P-47 toS-61; E-48 to S-62; A-49 to A-63; T-50 to G-64; N-51 to R-65; S-52 toH-66; S-53 to V-67; S-54 to R-68; S-55 to S-69; S-56 to Y-70; F-57 toN-71; S-58 to H-72; S-59 to L-73; P-60 to Q-74; S-61 to G-75; S-62 toD-76; A-63 to V-77; G-64 to R-78; R-65 to W-79; H-66 to R-80; V-67 toK-81; R-68 to L-82; S-69 to F-83; Y-70 to S-84; N-71 to F-85; H-72 toT-86; L-73 to K-87; Q-74 to Y-88; G-75 to F-89; D-76 to L-90; V-77 toK-91; R-78 to I-92; W-79 to E-93; R-80 to K-94; K-81 to N-95; L-82 toG-96; F-83 to K-97; S-84 to V-98; F-85 to S-99; T-86 to G-100; K-87 toT-101; Y-88 to K-102; F-89 to K-103; L-90 to E-104; K-91 to N-105; 1-92to C-106; E-93 to P-107; K-94 to Y-108; N-95 to S-109; G-96 to I-110;K-97 to L-111; V-98 to E-112; S-99 to I-113; G-100 to T-114; T-101 toS-115; K-102 to V-116; K-103 to E-117; E-104 to I-118; N-105 to G-119;C-106 to V-120; P-107 to V-121; Y-108 to A-122; S-109 to V-123; I-110 toK-124; L-111 to A-125;E-112to I-126;I-113 to N-127;T-114 to S-128; S-115to N-129; V-116 to Y-130; E-117 to Y-131; I-118 to L-132; G-119 toA-133; V-120 to M-134; V-121 to N-135; A-122 to K-136; V-123 to K-137;K-124 to G-138; A-125 to K-139; 1-126 to L-140; N-127 to Y-141; S-128 toG-142;N-129to S-143;Y-130to K-144;Y-131 to E-145;L-132 to F-146;A-133 toN-147; M-134 to N-148; N-135 to D-149; K-136 to C-150; K-137 to K-151;G-138 to L-152; K-139 to K-153; L-140 to E-154; Y-141 to R-155; G-142 toI-156; S-143 to E-157; K-144 to E-158; E-145 to N-159; F-146 to G-160;N-147 to Y-161; N-148 to N-162; D-149 to T-163; C-150 to Y-164; K-151 toA-165; L-152 to S-166; K-153 to F-167; E-154 to N-168; R-155 to W-169;I-156 to Q-170; E-157 to H-171; E-158 to N-172; N-159 to G-173; G-160 toR-174; Y-161 to Q-175; N-162 to M-176; T-163 to Y-177; Y-164 to V-178;A-165 to A-179; S-166 to L-180; F-167 to N-181; N-168 to G-182; W-169 toK-183; Q-170 to G-184; H-171 to A-185; N-172 to P-186; G-173 to R-187;R-174 to R-188; Q-175 to G-189; M-176 to Q-190; Y-177 to K-191; V-178 toT-192; A-179 to R-193; L-180 to R-194; N-181 to K-195; G-182 to N-196;K-183 to T-197; G-184 to S-198; A-185 to A-199; P-186 to H-200; R-187 toF-201; R-188 to L-202; G-189to P-203; Q-190 to M-204; K-191 to V-205;T-192 to V-206; R-193 to H-207; and/or R-194 to S-208 of SEQ ID NO:2.Polynucleotides encoding these polypeptide fragments are alsoencompassed by the invention.

Additional non-exclusive preferred antigenic epitopes include theantigenic epitopes disclosed herein, as well as portions thereof.Antigenic epitopes are useful, for example, to raise antibodies,including monoclonal antibodies, that specifically bind the epitope.Preferred antigenic epitopes include the antigenic epitopes disclosedherein, as well as any combination of two, three, four, five or more ofthese antigenic epitopes. Antigenic epitopes can be used as the targetmolecules in immunoassays. (See, for instance, Wilson et al., Cell37:767–778 (1984); Sutcliffe et al., Science 219:660–666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910–914; and Bittle et al., J. Gen. Virol.66:2347–2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910–914; and Bittle, F. J. et al., J.Gen. Virol. 66:2347–2354 (1985). Generally, animals may be immunizedwith free peptide; however, anti-peptide antibody titer may be boostedby coupling of the peptide to a macromolecular carrier, such as keyholelimpet hemacyanin (KLH) or tetanus toxoid. For instance, peptidescontaining cysteine may be coupled to carrier using a linker such asm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg peptide or carrier protein and Freund's adjuvant. Severalbooster injections may be needed, for instance, at intervals of abouttwo weeks, to provide a useful titer of anti-peptide antibody which canbe detected, for example, by ELISA assay using free peptide adsorbed toa solid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art. Forinstance, Geysen et al., supra, discloses a procedure for rapidconcurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art. For instance,the immunologically important epitope in the coat protein offoot-and-mouth disease virus was located by Geysen et al. with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁–C₇-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

As one of skill in the art will appreciate, KGF-2 polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84–86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric KGF-2 protein or proteinfragment alone (Fountoulakis et al., J Biochem 270:3958–3964 (1995)).

In accordance with the present invention, novel variants of KGF-2 arealso described. These can be produced by deleting or substituting one ormore amino acids of KGF-2. Natural mutations are called allelicvariations. Allelic variations can be silent (no change in the encodedpolypeptide) or may have altered amino acid sequence.

In order to attempt to improve or alter the characteristics of nativeKGF-2, protein engineering may be employed. Recombinant DNA technologyknown to those skilled in the art can be used to create novelpolypeptides. Muteins and deletions can show, e.g., enhanced activity orincreased stability. In addition, they could be purified in higher yieldand show better solubility at least under certain purification andstorage conditions. Set forth below are examples of mutations that canbe constructed.

The KGF-2 polypeptides of the invention may be in monomers or multimers(i.e., dimers, trimers, tetramers and higher multimers). Accordingly,the present invention relates to monomers and multimers of the KGF-2polypeptides of the invention, their preparation, and compositions(preferably, Therapeutics) containing them. In specific embodiments, thepolypeptides of the invention are monomers, dimers, trimers ortetramers. In additional embodiments, the multimers of the invention areat least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides corresponding to the amino acid sequence of SEQ ID NO:2 orencoded by the cDNA contained in the deposited clone (includingfragments, variants, splice variants, and fusion proteins, correspondingto these as described herein). These homomers may contain KGF-2polypeptides having identical or different amino acid sequences. In aspecific embodiment, a homomer of the invention is a multimer containingonly KGF-2 polypeptides having an identical amino acid sequence. Inanother specific embodiment, a homomer of the invention is a multimercontaining KGF-2 polypeptides having different amino acid sequences. Inspecific embodiments, the multimer of the invention is a homodimer(e.g., containing KGF-2 polypeptides having identical or different aminoacid sequences) or a homotrimer (e.g., containing KGF-2 polypeptideshaving identical and/or different amino acid sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the KGF-2 polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the KGF-2 polypeptides of theinvention. Such covalent associations may involve one or more amino acidresidues contained in the polypeptide sequence (e.g., that recited inSEQ ID NO:2, or contained in the polypeptides encoded by the cloneHPRCC57 or the clone contained in ATCC® Deposit No. 75977 or 75901). Inone instance, the covalent associations are cross-linking betweencysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a KGF-2 fusion protein. In oneexample, covalent associations are between the heterologous sequencecontained in a fusion protein of the invention (see, e.g., U.S. Pat. No.5,478,925). In a specific example, the covalent associations are betweenthe heterologous sequence contained in a KGF-2-Fc fusion protein of theinvention (as described herein). In another specific example, covalentassociations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

In another example, proteins of the invention are associated byinteractions between FLAG® polypeptide sequence contained in fusionproteins of the invention containing FLAG® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in FLAG® fusion proteins of the invention and anti FLAG®antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hyrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Polynucleotide and Polypeptide Fragments

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having, for example, the nucleotide sequence ofthe deposited cDNA (clone HPRCC57), a nucleotide sequence encoding thepolypeptide sequence encoded by the deposited cDNA, a nucleotidesequence encoding the polypeptide sequence depicted in FIGS. 1A–1C (SEQID NO:2), the nucleotide sequence shown in FIGS. 1A–1C (SEQ ID NO:1), orthe complementary strand thereto, is intended fragments at least 15 nt,and more preferably at least about 20 nt, still more preferably at least30 nt, and even more preferably, at least about 40, 50, 100, 150, 200,250, 300, 325, 350, 375, 400, 450, 500, 550, or 600 nt in length. Thesefragments have numerous uses that include, but are not limited to,diagnostic probes and primers as discussed herein. Of course, largerfragments, such as those of 501–1500 nt in length are also usefulaccording to the present invention as are fragments corresponding tomost, if not all, of the nucleotide sequences of the deposited cDNA(clone HPRCC57) or as shown in FIGS. 1A–1C (SEQ ID NO:1). By a fragmentat least 20 nt in length, for example, is intended fragments whichinclude 20 or more contiguous bases from, for example, the nucleotidesequence of the deposited cDNA, or the nucleotide sequence as shown inFIGS. 1A–1C (SEQ ID NO:1).

Moreover, representative examples of KGF-2 polynucleotide fragmentsinclude, for example, fragments having a sequence from about nucleotidenumber 1–50, 51–100, 101–150, 151–200, 201–250, 251–300, 301–350,351–400, 401–450, 451–500, 501–550, 551–600, 651–700, 701–750, 751–800,800–850, 851–900, 901–950, 951–1000, 1001–1050, 1051–1100, 1101–1150,1151–1200, 1201–1250, 1251–1300, 1301–1350, 1351–1400, 1401–1450,1451–1500, 1501–1550, 1551–1600, 1601–1650, 1651–1700, 1701–1750,1751–1800, 1801–1850, 1851–1900, 1901–1950, 1951–2000, and/or 2001 tothe end of SEQ ID NO:1 or the complementary strand thereto, or the cDNAcontained in the deposited clone. In this context “about” includes theparticularly recited ranges, larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates a KGF-2 functional activity. By apolypeptide demonstrating a KGF-2 “functional activity” is meant, apolypeptide capable of displaying one or more known functionalactivities associated with a full-length (complete) KGF-2 protein. Suchfunctional activities include, but are not limited to, biologicalactivity, antigenicity [ability to bind (or compete with a KGF-2polypeptide for binding) to an anti-KGF-2 antibody], immunogenicity(ability to generate antibody which binds to a KGF-2 polypeptide),ability to form multimers with KGF-2 polypeptides of the invention, andability to bind to a receptor or ligand for a KGF-2 polypeptide.

The functional activity of KGF-2 polypeptides, and fragments, variantsderivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length KGF-2 polypeptide for binding toanti-KGF-2 antibody, various immunoassays known in the art can be used,including but not limited to, competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, where a KGF-2 ligand is identified, or theability of a polypeptide fragment, variant or derivative of theinvention to multimerize is being evaluated, binding can be assayed,e.g., by means well-known in the art, such as, for example, reducing andnon-reducing gel chromatography, protein affinity chromatography, andaffinity blotting. See generally, Phizicky, E. et al., Microbiol. Rev.59:94–123 (1995). In another embodiment, physiological correlates ofKGF-2 binding to its substrates (signal transduction) can be assayed.

In addition, assays described herein (see Examples) and otherwise knownin the art may routinely be applied to measure the ability of KGF-2polypeptides and fragments, variants derivatives and analogs thereof toelicit KGF-2 related biological activity (either in vitro or in vivo).Other methods will be known to the skilled artisan and are within thescope of the invention.

The present invention is further directed to fragments of the KGF-2polypeptide described herein. By a fragment of an isolated the KGF-2polypeptide, for example, encoded by the deposited cDNA (clone HPRCC57),the polypeptide sequence encoded by the deposited cDNA, the polypeptidesequence depicted in FIGS. 1A–1C (SEQ ID NO:2), is intended to encompasspolypeptide fragments contained in SEQ ID NO:2 or encoded by the cDNAcontained in the deposited clone. Protein fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments from about amino acid number1–20, 21–40, 41–60, 61–80, 81–100, 102–120, 121–140, 141–160, 161–180,181–200, 201–220, 221–240, 241–260, 261–280, or 281 to the end of thecoding region. Moreover, polypeptide fragments can be at least 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids inlength. In this context “about” includes the particularly recitedranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, ateither extreme or at both extremes.

Even if deletion of one or more amino acids from the N-terminus of aprotein results in modification of loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities, ability to multimerize, ability to bind KGF-2 ligand) maystill be retained. For example, the ability of shortened KGF-2 muteinsto induce and/or bind to antibodies which recognize the complete ormature forms of the polypeptides generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the N-terminus. Whether a particular polypeptidelacking N-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thatan KGF-2 mutein with a large number of deleted N-terminal amino acidresidues may retain some biological or immunogenic activities. In fact,peptides composed of as few as six KGF-2 amino acid residues may oftenevoke an immune response.

Accordingly, polypeptide fragments include the secreted KGF-2 protein aswell as the mature form. Further preferred polypeptide fragments includethe secreted KGF-2 protein or the mature form having a continuous seriesof deleted residues from the amino or the carboxy terminus, or both. Forexample, any number of amino acids, ranging from 1–60, can be deletedfrom the amino terminus of either the secreted KGF-2 polypeptide or themature form. Similarly, any number of amino acids, ranging from 1–30,can be deleted from the carboxy terminus of the secreted KGF-2 proteinor mature form. Furthermore, any combination of the above amino andcarboxy terminus deletions are preferred. Similarly, polynucleotidefragments encoding these KGF-2 polypeptide fragments are also preferred.

Particularly, N-terminal deletions of the KGF-2 polypeptide can bedescribed by the general formula m-208, where m is an integer from 2 to207, where m corresponds to the position of the amino acid residueidentified in SEQ ID NO:2. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of residues of W-2 to S-208; K-3to S-208; W-4 to S-208; I-5 to S-208; L-6 to S-208; T-7 to S-208; H-8 toS-208; C-9 to S-208; A-10 to S-208; S-11 to S-208; A-12 to S-208; F-13to S-208; P-14 to S-208; H-15 to S-208; L-16 to S-208; P-17 to S-208;G-18 to S-208; C-19 to S-208; C-20 to S-208; C-21 to S-208; C-22 toS-208; C-23 to S-208; F-24 to S-208; L-25 to S-208; L-26 to S-208; L-27to S-208; F-28 to S-208; L-29 to S-208; V-30 to S-208; S-31 to S-208;S-32 to S-208; V-33 to S-208; P-34 to S-208; V-35 to S-208; T-36 toS-208; C-37 to S-208; Q-38 to S-208; A-39 to S-208; L-40 to S-208; G-41to S-208; Q-42 to S-208; D-43 to S-208; M-44 to S-208; V-45 to S-208;S-46 to S-208; P-47 to S-208; E-48 to S-208; A-49 to S-208; T-50 toS-208; N-51 to S-208; S-52 to S-208; S-53 to S-208; S-54 to S-208; S-55to S-208; S-56 to S-208; F-57 to S-208; S-58 to S-208; S-59 to S-208;P-60 to S-208; S-61 to S-208; S-62 to S-208; A-63 to S-208; G-64 toS-208; R-65 to S-208; H-66 to S-208; V-67 to S-208; R-68 to S-208; S-69to S-208; Y-70 to S-208; N-71 to S-208; H-72 to S-208; L-73 to S-208;Q-74 to S-208; G-75 to S-208; D-76 to S-208; V-77 to S-208; R-78 toS-208; W-79 to S-208; R-80 to S-208; K-81 to S-208; L-82 to S-208; F-83to S-208; S-84 to S-208; F-85 to S-208; T-86 to S-208; K-87 to S-208;Y-88 to S-208; F-89 to S-208; L-90 to S-208; K-91 to S-208; I-92 toS-208; E-93 to S-208; K-94 to S-208; N-95 to S-208; G-96 to S-208; K-97to S-208; V-98 to S-208; S-99 to S-208; G-100 to S-208; T-101 to S-208;K-102 to S-208; K-103 to S-208; E-104 to S-208; N-105 to S-208; C-106 toS-208; P-107 to S-208; Y-108 to S-208; S-109 to S-208; I-110 to S-208;L-111 to S-208; E-112 to S-208; I-113 to S-208; T-114 to S-208; S-115 toS-208; V-116 to S-208; E-117 to S-208; I-118 to S-208; G-119 to S-208;V-120 to S-208; V-121 to S-208; A-122 to S-208; V-123 to S-208; K-124 toS-208; A-125 to S-208; I-126 to S-208; N-127 to S-208; S-128 to S-208;N-129 to S-208; Y-130 to S-208; Y-131 to S-208; L-132 to S-208; A-133 toS-208; M-134 to S-208; N-135 to S-208; K-136 to S-208; K-137 to S-208;G-138 to S-208; K-139 to S-208; L-140 to S-208; Y-141 to S-208; G-142 toS-208; S-143 to S-208; K-144 to S-208; E-145 to S-208; F-146 to S-208;N-147 to S-208; N-148 to S-208; D-149 to S-208; C-150 to S-208; K-151 toS-208; L-152 to S-208; K-153 to S-208; E-154 to S-208; R-155 to S-208;I-156 to S-208; E-157 to S-208; E-158 to S-208; N-159 to S-208; G-160 toS-208; Y-161 to S-208; N-162 to S-208; T-163 to S-208; Y-164 to S-208;A-165 to S-208; S-166 to S-208; F-167 to S-208; N-168 to S-208; W-169 toS-208; Q-170 to S-208; H-171 to S-208; N-172 to S-208; G-173 to S-208;R-174 to S-208; Q-175 to S-208; M-176 to S-208; Y-177 to S-208; V-178 toS-208; A-179 to S-208; L-180 to S-208; N-181 to S-208; G-182 to S-208;K-183 to S-208; G-184 to S-208; A-185 to S-208; P-186 to S-208; R-187 toS-208; R-188 to S-208; G-189 to S-208; Q-190 to S-208; K-191 to S-208;T-192 to S-208; R-193 to S-208; R-194 to S-208; K-195 to S-208; N-196 toS-208; T-197 to S-208; S-198 to S-208; A-199 to S-208; H-200 to S-208;F-201 to S-208; L-202 to S-208; P-203 to S-208; of SEQ ID NO:2.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Particularly preferred are fragments comprising or consisting of:S69-S208; A63-S208; Y70-S208; V77-S208; E93-S208; E104-S208; V123-S208;G138-S208; R80-S208; A39-S208; S69-V178; S69-G173; S69-R188; S69-S198;S84-S208; V98-S208; A63-N162; S69-N162; and M35-N162.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, ability to bindKGF-2 ligand) may still be retained. For example the ability of theshortened KGF-2 mutein to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptide generally willbe retained when less than the majority of the residues of the completeor mature polypeptide are removed from the C-terminus. Whether aparticular polypeptide lacking C-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that an KGF-2 mutein with a large number ofdeleted C-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixKGF-2 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the KGF-2 polypeptide shown in FIGS. 1A–1C (SEQ ID NO:2), asdescribed by the general formula 1-n, where n is an integer from 2 to207, where n corresponds to the position of amino acid residueidentified in SEQ ID NO:2. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of residues M-1 to H-207; M-1 toV-206; M-1 to V-205; M-1 to M-204; M-1 to P-203; M-1 to L-202; M-1 toF-201; M-1 to H-200; M-1 to A-199; M-1 to S-198; M-1 to T-197; M-1 toN-196; M-1 to K-195; M-1 to R-194; M-1 to R-193; M-1 to T-192; M-1 toK-191; M-1 to Q-190; M-1 to G-189; M-1 to R-188; M-1 to R-187; M-1 toP-186; M-1 to A-185; M-1 to G-184; M-1 to K-183; M-1 to G-182; M-1 toN-181; M-1 to L-180; M-1 to A-179; M-1 to V-178; M-1 to Y-177; M-1 toM-176; M-1 to Q-175; M-1 to R-174; M-1 to G-173; M-1 to N-172; M-1 toH-171; M-1 to Q-170; M-1 to W-169; M-1 to N-168; M-1 to F-167; M-1 toS-166; M-1 to A-165; M-1 to Y-164; M-1 to T-163; M-1 to N-162; M-1 toY-161; M-1 to G-160; M-1 to N-159; M-1 to E-158; M-1 to E-157; M-1 toI-156; M-1 to R-155; M-1 to E-154; M-1 to K-153; M-1 to L-152; M-1 toK-151; M-1 to C-150; M-1 to D-149; M-1 to N-148; M-1 to N-147; M-1 toF-146; M-1 to E-145; M-1 to K-144; M-1 to S-143; M-1 to G-142; M-1 toY-141; M-1 to L-140; M-1 to K-139; M-1 to G-138; M-1 to K-137; M-1 toK-136; M-1 to N-135; M-1 to M-134; M-1 to A-133; M-1 to L-132; M-1 toY-131; M-1 to Y-130; M-1 to N-129; M-1 to S-128; M-1 to N-127; M-1 toI-126;M-1 to A-125; M-1 to K-124; M-1 to V-123; M-1 to A-122; M-1 toV-121; M-1 to V-120; M-1 to G-119; M-1 to I-118; M-1 to E-117; M-1 toV-116; M-1 to S-115; M-1 to T-114; M-1 to I-113; M-1 to E-112; M-1 toL-11; M-1 to I-110; M-1 to S-109; M-1 to Y-108; M-1 to P-107; M-1 toC-106; M-1 to N-105; M-1 to E-104; M-1 to K-103; M-1 to K-102; M-1 toT-101; M-1 to G-100; M-1 to S-99; M-1 to V-98; M-1 to K-97; M-1 to G-96;M-1 to N-95; M-1 to K-94; M-1 to E-93; M-1 to I-92; M-1 to K-91; M-1 toL-90; M-1 to F-89; M-1 to Y-88; M-1 to K-87; M-1 to T-86; M-1 to F-85;M-1 to S-84; M-1 to F-83; M-1 to L-82; M-1 to K-81; M-1 to R-80; M-1 toW-79; M-1 to R-78; M-1 to V-77; M-1 to D-76; M-1 to G-75; M-1 to Q-74;M-1 to L-73; M-1 to H-72; M-1 to N-71; M-1 to Y-70; M-1 to S-69; M-1 toR-68; M-1 to V-67; M-1 to H-66; M-1 to R-65; M-1 to G-64; M-1 to A-63;M-1 to S-62; M-1 to S-61; M-1 to P-60; M-1 to S-59; M-1 to S-58; M-1 toF-57; M-1 to S-56; M-1 to S-55; M-1 to S-54; M-1 to S-53; M-0.1 to S-52;M-1 to N-51; M-1 to T-50; M-1 to A-49; M71 to E-48; M-1 to P-47; M-1 toS-46; M-1 to V-45; M-1 to M-44; M-1 to D-43; M-1 to Q-42; M-1 to G-41;M-1 to L-40; M-1 to A-39; M-1 to Q-38; M-1 to C-37; M-1 to T-36; M-1 toV-35; M-1 to P-34; M-1 to V-33; M-1 to S-32; M-1 to S-31; M-1 to V-30;M-1 to L-29; M-1 to F-28; M-1 to L-27; M-1 to L-26; M-1 to L-25; M-1 toF-24; M-1 to C-23; M-1 to C-22; M-1 to C-21; M-1 to C-20; M-1 to C-19;M-1 to G-18; M-1 to P-17; M-1 to L-16; M-1 to H-15; M-1 to P-14; M-1 toF-13; M-1 to A-12; M-1 to S-11; M-1 to A-10; M-1 to C-9; M-1 to H-8; M-1to T-7; of SEQ ID NO:2. Polynucleotides encoding these polypeptides arealso encompassed by the invention.

Likewise, C-terminal deletions of the KGF-2 polypeptide of the inventionshown

as SEQ ID NO:2 include polypeptides comprising the amino acid sequenceof residues: S-69 to H-207; S-69 to V-206; S-69 to V-205; S-69 to M-204;S-69 to P-203; S-69 to L-202; S-69 to F-201; S-69 to H-200; S-69 toA-199; S-69 to S-198; S-69 to T-197; S-69 to N-196; S-69 to K-195; S-69to R-194; S-69 to R-193; S-69 to T-192; S-69 to K-191; S-69 to Q-190;S-69 to G-189; S-69 to R-188; S-69 to R-187; S-69 to P-186; S-69 toA-185; S-69 to G-184; S-69 to K-183; S-69 to G-182; S-69 to N-181; S-69to L-180; S-69 to A-179; S-69 to V-178; S-69 to Y-177; S-69 to M-176;S-69 to Q-175; S-69 to R-174; S-69 to G-173; S-69 to N-172; S-69 toH-171; S-69 to Q-170; S-69 to W-169; S-69 to N-168; S-69 to F-167; S-69to S-166; S-69 to A-165; S-69 to Y-164; S-69 to T-163; S-69 to N-162;S-69 to Y-161; S-69 to G-160; S-69 to N-159; S-69 to E-158; S-69 toE-157; S-69 to I-156; S-69 to R-155; S-69 to E-154; S-69 to K-153; S-69to L-152; S-69 to K-151; S-69 to C-150; S-69 to D-149; S-69 to N-148;S-69 to N-147; S-69 to F-146; S-69 to E-145; S-69 to K-144; S-69 toS-143; S-69 to G-142; S-69 to Y-141; S-69 to L-140; S-69 to K-139; S-69to G-138; S-69 to K-137; S-69 to K-136; S-69 to N-135; S-69 to M-134;S-69 to A-133; S-69 to L-132; S-69 to Y-131; S-69 to Y-130; S-69 toN-129; S-69 to S-128; S-69 to N-127; S-69 to I-126; S-69 to A-125; S-69to K-124; S-69 to V-123; S-69 to A-122; S-69 to V-121; S-69 to V-120;S-69 to G-119; S-69 to I-118; S-69 to E-117; S-69 to V-116; S-69 toS-115; S-69 to T-114; S-69 to I-i 13; S-69 to E-112; S-69 to L-111; S-69to I-110; S-69 to S-109; S-69 to Y-108; S-69 to P-107; S-69 to C-106;S-69 to N-105; S-69 to E-104; S-69 to K-103; S-69 to K-102; S-69 toT-101; S-69 to G-100; S-69 to S-99; S-69 to V-98; S-69 to K-97; S-69 toG-96; S-69 to N-95; S-69 to K-94; S-69 to E-93; S-69 to I-92; S-69 toK-91; S-69 to L-90; S-69 to F-89; S-69 to Y-88; S-69 to K-87; S-69 toT-86; S-69 to F-85; S-69 to S-84; S-69 to F-83; S-69 to L-82; S-69 toK-81; S-69 to R-80; S-69 to W-79; S-69 to R-78; S-69 to V-77; S-69 toD-76; S-69 to G-75; of SEQ ID NO:2.

In addition, any of the above listed N- or C-terminal deletions can becombined to produce a N- and C-terminal deleted KGF-2 polypeptide. Theinvention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues m-n of SEQ ID NO:2, where n and mare integers as described above. In addition, N- or C-terminal deletionmutants may also contain site specific amino acid substitutions.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete KGF-2 amino acid sequenceencoded by the cDNA clone contained in ATCC® Deposit No. 75977, wherethis portion excludes any integer of amino acid residues from 1 to about198 amino acids from the amino terminus of the complete amino acidsequence encoded by the cDNA clone contained in ATCC® Deposit No. 75977,or any integer of amino acid residues from 1 to about 198 amino acidsfrom the carboxy terminus, or any combination of the above aminoterminal and carboxy terminal deletions, of the complete amino acidsequence encoded by the cDNA clone contained in ATCC® Deposit No. 75977.Polynucleotides encoding all of the above deletion mutant polypeptideforms also are provided.

The present application is also directed to proteins containingpolypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%or. 99% identical to the KGF-2 polypeptide sequence set forth hereinm-n. In preferred embodiments, the application is directed to proteinscontaining polypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,97%, 98% or 99% identical to polypeptides having the amino acid sequenceof the specific KGF-2- and C-terminal deletions recited herein.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Among the especially preferred fragments of the invention are fragmentscharacterized by structural or functional attributes of KGF-2. Suchfragments include amino acid residues that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet-forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., containing four or morecontiguous amino acids having an antigenic index of greater than orequal to 1.5, as identified using the default parameters of theJameson-Wolf program) of complete (i.e., full-length) KGF-2 (SEQ IDNO:2). Certain preferred regions are those set out in FIG. 4 andinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence depicted in FIGS.1A–1C (SEQ ID NO:2), such preferred regions include; Garnier-Robsonpredicted alpha-regions, beta-regions, turn-regions, and coil-regions;Chou-Fasman predicted alpha-regions, beta-regions, turn-regions, andcoil-regions; Kyte-Doolittle predicted hydrophilic and hydrophobicregions; Eisenberg alpha and beta amphipathic regions; Eminisurface-forming regions; and Jameson-Wolf high antigenic index regions,as predicted using the default parameters of these computer programs.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of KGF-2. Preferred embodiments of the inventionin this regard include fragments that comprise alpha-helix andalpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheetforming regions (“beta-regions”), turn and turn-forming regions(“turn-regions”), coil and coil-forming regions (“coil-regions”),hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions andhigh antigenic index regions of KGF-2.

The data representing the structural or functional attributes of KGF-2set forth in FIGS. 1A–1C and/or Table I, as described above, wasgenerated using the various modules and algorithms of the DNA*STAR seton default parameters. In a preferred embodiment, the data presented incolumns VIII, IX, XIII, and XIV of Table I can be used to determineregions of KGF-2 which exhibit a high degree of potential forantigenicity. Regions of high antigenicity are determined from the datapresented in columns VIII, IX, XIII, and/or IV by choosing values whichrepresent regions of the polypeptide which are likely to be exposed onthe surface of the polypeptide in an environment in which antigenrecognition may occur in the process of initiation of an immuneresponse.

Certain preferred regions in these regards are set out in FIG. 4, butmay, as shown in Table I, be represented or identified by using tabularrepresentations of the data presented in FIG. 4. The DNA*STAR computeralgorithm used to generate FIG. 4 (set on the original defaultparameters) was used to present the data in FIG. 4 in a tabular format(See Table 1). The tabular format of the data in FIG. 4 may be used toeasily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 4 and in Table Iinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS.1A–1C. As set out in FIG. 4 and in Table I, such preferred regionsinclude Garnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions,Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenbergalpha- and beta-amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf regions of high antigenicindex. The columns are labeled with the headings “Res”, “Position”, andRoman Numerals I–XIV. The column headings refer to the followingfeatures of the amino acid sequence presented in FIG. 3, and Table I:“Res”: amino acid residue of SEQ ID NO:2 and FIGS. 1A and 1B;“Position”: position of the corresponding residue within SEQ ID NO:2 andFIGS. 1A and 1B; I: Alpha, Regions—Garnier-Robson; II: Alpha,Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta,Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn,Regions—Chou-Fasman; VII: Coil, Regions—Garnier-Robson; VIII:Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods;X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, AmphipathicRegions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: AntigenicIndex—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.

TABLE I Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met1 A A . . . . . −0.08 0.73 * . . −0.60 0.82 Trp 2 A A . . . . . −0.500.99 * . . −0.60 0.45 Lys 3 A A . . . . . −0.42 1.24 * . . −0.60 0.29Trp 4 A A . . . . . −0.07 1.30 * . . −0.60 0.42 Ile 5 A A . . . . .−0.34 1.19 * . . −0.60 0.55 Leu 6 A A . . . . . −0.33 0.84 * . . −0.600.15 Thr 7 . A B . . . . −0.34 1.34 * . . −0.60 0.14 His 8 . A . . T . .−0.98 0.81 * . . −0.20 0.27 Cys 9 . A . . T . . −1.39 0.63 . . . −0.200.33 Ala 10 . A . . T . . −0.71 0.73 * . . −0.20 0.20 Ser 11 . A . . T .. 0.07 0.67 * . . −0.20 0.23 Ala 12 . A . . T . . −0.43 0.67 * . . −0.200.57 Phe 13 . A B . . . . −0.61 0.79 . . . −0.60 0.47 Pro 14 . . . . T .. −0.29 0.71 . . . 0.00 0.54 His 15 . . . . T . . −0.37 0.76 . . . 0.000.53 Leu 16 . . . . T T . −0.73 0.83 . . . 0.20 0.33 Pro 17 . . . . T T. −0.81 0.61 . . . 0.20 0.11 Gly 18 . . . . T T . −0.78 0.76 . . . 0.200.04 Cys 19 . . . . T T . −1.23 0.83 . . . 0.20 0.03 Cys 20 . . . . T T. −1.90 0.71 . . . 0.20 0.01 Cys 21 . . B . . T . −1.90 1.07 . . . −0.200.01 Cys 22 . . B . . T . −2.50 1.33 . . . −0.20 0.01 Cys 23 . . B . . T. −2.97 1.44 . . . −0.20 0.02 Phe 24 . . B B . . . −3.00 1.56 . . .−0.60 0.03 Leu 25 . . B B . . . −3.14 1.77 . . . −0.60 0.05 Leu 26 . . BB . . . −3.33 1.89 . . . −0.60 0.08 Leu 27 . . B B . . . −2.97 1.96 . .. −0.60 0.07 Phe 28 . . B B . . . −2.60 1.56 . . . −0.60 0.11 Leu 29 . .B B . . . −2.76 1.26 . . . −0.60 0.18 Val 30 . . B B . . . −2.16 1.21 .. . −0.60 0.16 Ser 31 . . . B T . . −2.20 0.96 . . . −0.20 0.29 Ser 32 .. . B . . C −1.70 0.81 . . . −0.40 0.26 Val 33 . . B B . . . −1.67 0.61. . . −0.60 0.51 Pro 34 . . B B . . . −0.86 0.54 . * . −0.60 0.20 Val 35. . B B . . . −0.59 0.56 . . . −0.60 0.26 Thr 36 . . B B . . . −1.100.67 . * . −0.60 0.36 Cys 37 . . B B . . . −1.14 0.71 . * . −0.60 0.19Gln 38 . . B B . . . −0.29 0.71 . * . −0.60 0.25 Ala 39 . . B B . . .−0.08 0.47 . . . −0.60 0.30 Leu 40 . . B B . . . 0.18 −0.01 . . . 0.300.95 Gly 41 . . B . . T . −0.37 0.03 . . F 0.25 0.54 Gln 42 . . B . . T. 0.00 0.27 * . F 0.25 0.40 Asp 43 . . B . . T . −0.21 0.16 . . F 0.250.65 Met 44 . . B . . T . 0.38 −0.10 . . F 1.00 1.01 Val 45 . . B . . .. 0.60 −0.53 . . . 0.95 1.01 Ser 46 . . B . . T . 0.63 −0.43 . . F 0.850.61 Pro 47 . . B . . T . 0.63 0.06 . . F 0.49 0.89 Glu 48 A . B . . T .0.33 −0.16 . . F 1.48 1.93 Ala 49 A . . . . T . 0.63 −0.41 . . F 1.721.93 Thr 50 A . . . . . . 1.19 −0.41 . . F 1.76 1.67 Asn 51 . . . . . TC 1.19 −0.46 . . F 2.40 1.29 Ser 52 . . . . . T C 1.10 −0.07 . . F 2.161.72 Ser 53 . . . . . T C 0.40 −0.19 . . F 1.92 1.59 Ser 54 . . . . T T. 0.69 0.11 . . F 1.13 0.86 Ser 55 . . . . . T C 0.70 0.10 . . F 0.690.86 Ser 56 . . . . T T . 0.49 0.10 . . F 0.65 0.86 Phe 57 . . . . T T .0.49 0.14 . . F 0.65 0.99 Ser 58 . . . . . T C 0.49 0.14 . . F 0.69 0.99Ser 59 . . . . . T C 0.20 0.14 . . F 0.93 0.99 Pro 60 . . . . . T C 0.160.26 * . F 1.32 1.15 Ser 61 . . . . . T C 0.57 −0.10 * . F 2.01 0.85 Ser62 . . . . . T C 1.23 −0.49 * . F 2.40 1.25 Ala 63 . . . . . . C 0.68−0.37 * . F 1.96 1.10 Gly 64 . . B . . . . 1.09 −0.16 * . F 1.37 0.61Arg 65 . . B . . . . 1.00 −0.54 * . F 1.43 0.89 His 66 . . B . . . .1.06 −0.54 * . . 1.19 1.18 Val 67 . . B . . . . 1.36 −0.29 * . . 0.651.86 Arg 68 . . B . . T . 1.91 −0.31 * . . 0.85 1.53 Ser 69 . . B . . T. 1.44 0.19 * * . 0.25 1.53 Tyr 70 . . B . . T . 1.33 0.37 * * . 0.251.70 Asn 71 . . . . T T . 1.02 0.13 * * . 0.65 1.50 His 72 . . . . . . C1.88 0.56 * * . −0.05 1.11 Leu 73 . . . . . T C 0.91 0.17 * * . 0.451.18 Gln 74 . . B . . T . 1.32 0.06 * * F 0.25 0.55 Gly 75 . . B . . T .1.28 −0.34 . * F 0.85 0.79 Asp 76 . . B . . T . 1.39 0.07 . * F 0.401.00 Val 77 . . B B . . . 1.47 −0.61 . * F 0.90 1.13 Arg 78 . . B B . .. 1.47 −1.01 * * . 0.75 2.29 Trp 79 . . B B . . . 0.77 −0.76 * * . 0.751.13 Arg 80 . . B B . . . 0.81 0.03 * * . −0.15 1.32 Lys 81 . . B B . .. 0.11 −0.23 . * . 0.30 0.90 Leu 82 . . B B . . . 0.66 0.56 * * . −0.600.74 Phe 83 . . B B . . . 0.59 0.13 * * . −0.30 0.55 Ser 84 . . B B . .. 0.63 0.13 * . . −0.30 0.55 Phe 85 A . . B . . . −0.18 0.89 * . . −0.451.04 Thr 86 A . . B . . . −1.03 0.99 * . . −0.45 1.04 Lys 87 A A . B . .. −0.18 0.89 * * . −0.60 0.64 Tyr 88 A A . B . . . −0.37 0.50 * * .−0.45 1.48 Phe 89 A A . B . . . −0.07 0.40 * . . −0.30 0.72 Leu 90 A A .B . . . 0.68 −0.09 * * . 0.30 0.62 Lys 91 A A . B . . . 0.99 −0.09 * * F0.45 0.79 Ile 92 A A . . . . . 0.60 −0.44 * * F 0.60 1.48 Glu 93 A . . .. T . 0.89 −0.80 * * F 1.30 1.77 Lys 94 A . . . . T . 0.73 −1.49 * * F1.30 1.77 Asn 95 A . . . . T . 1.24 −0.84 . * F 1.30 1.88 Gly 96 A . . .. T . 0.86 −1.14 * * F 1.64 1.45 Lys 97 A . . . . . . 1.43 −0.71 * * F1.63 0.72 Val 98 A . . . . . . 1.48 −0.23 . * F 1.67 0.64 Ser 99 . . . .. . C 1.48 −0.63 . . F 2.66 1.30 Gly 100 . . . . T T . 1.48 −1.06 . * F3.40 1.30 Thr 101 . . B . . T . 1.82 −1.06 . * F 2.66 3.04 Lys 102 . . B. . T . 1.11 −1.30 . * F 2.49 3.65 Lys 103 . . . . T T . 1.76 −1.11 . .F 2.72 1.98 Glu 104 . . . . T . . 1.81 −1.11 . . F 2.35 2.12 Asn 105 . .. . T . . 1.86 −0.84 . . F 2.18 1.66 Cys 106 . . B . . T . 1.28 −0.46 .. . 1.70 1.11 Pro 107 . . . . T T . 0.42 0.23 . . . 1.18 0.45 Tyr 108 .. . . T T . 0.38 0.91 . . . 0.71 0.23 Ser 109 . . B . . T . −0.51 0.51 *. . 0.14 0.75 Ile 110 . . B B . . . −0.82 0.63 * . . −0.43 0.34 Leu 111. . B B . . . −0.46 0.69 . . . −0.60 0.31 Glu 112 . . B B . . . −1.100.31 . . . −0.30 0.31 Ile 113 . . B B . . . −0.86 0.57 . . . −0.60 0.33Thr 114 . . B B . . . −1.44 −0.11 . . F 0.45 0.69 Ser 115 . . B B . . .−0.90 −0.11 . . F 0.45 0.28 Val 116 A . . B . . . −0.94 0.31 . . . −0.300.40 Glu 117 A . . B . . . −1.80 0.27 . . . −0.30 0.20 Ile 118 A . . B .. . −1.50 0.43 . . . −0.60 0.11 Gly 119 A . . B . . . −2.04 0.54 . * .−0.60 0.15 Val 120 A . . B . . . −1.70 0.54 . * . −0.60 0.07 Val 121 A .. B . . . −1.43 0.54 * . . −0.60 0.19 Ala 122 A . . B . . . −2.32 0.36 *. . −0.30 0.19 Val 123 . . B B . . . −1.43 0.61 * . . −0.60 0.18 Lys 124. . B B . . . −1.39 0.37 . . . −0.30 0.39 Ala 125 . . B . . . . −0.530.11 . . . −0.10 0.52 Ile 126 . . B . . . . 0.08 0.01 * . . 0.05 1.13Asn 127 . . B . . T . 0.42 0.13 * . F 0.25 0.88 Ser 128 . . B . . T .0.47 0.89 * . F 0.10 1.37 Asn 129 . . B . . T . −0.17 1.07 * . . −0.051.61 Tyr 130 . . B . . T . −0.18 0.89 . * . −0.05 1.01 Tyr 131 A A . . .. . 0.71 1.10 . * . −0.60 0.75 Leu 132 A A . . . . . 0.76 1.11 . . .−0.60 0.75 Ala 133 A A . . . . . 1.10 0.71 . . . −0.60 0.95 Met 134 A A. . . . . 0.76 −0.04 . * . 0.45 1.22 Asn 135 A . . . . T . 1.04 −0.37. * . 0.85 1.46 Lys 136 A . . . . T . 0.48 −1.06 . * F 1.30 2.89 Lys 137A . . . . T . 1.04 −0.87 . * F 1.30 2.41 Gly 138 A . . . . T . 1.29−0.73 . * F 1.30 2.34 Lys 139 A . . . . . . 1.59 −0.70 * * F 1.10 1.16Leu 140 . . B . . . . 1.63 −0.31 . * F 0.65 0.78 Tyr 141 . . B . . T .1.59 −0.31 . * F 1.00 1.57 Gly 142 . . B . . T . 0.84 −0.74 . * F 1.301.36 Ser 143 . . B . . T . 1.19 0.04 . * F 0.40 1.43 Lys 144 . . B . . T. 1.14 −0.24 . * F 1.00 1.47 Glu 145 A . . . . . . 1.96 −0.60 * F 1.102.38 Phe 146 A . . . . . . 1.53 −1.03 * * F 1.10 2.97 Asn 147 A . . . .T . 1.92 −0.84 * * F 1.15 0.80 Asn 148 A . . . . T . 1.41 −0.84 . * F1.15 0.92 Asp 149 A . . . . T . 1.41 −0.16 . * F 0.85 0.88 Cys 150 A . .. . T . 1.41 −0.94 * * F 1.30 1.09 Lys 151 A A . . . . . 2.22 −1.34 * *F 0.90 1.17 Leu 152 A A . . . . . 1.33 −1.74 * * F 0.90 1.37 Lys 153 A A. . . . . 1.33 −1.06 * * F 0.90 1.80 Glu 154 A A . . . . . 1.33−1.63 * * F 0.90 1.56 Arg 155 A A . . . . . 2.00 −1.63 * * F 0.90 3.27Ile 156 A A . . . . . 1.61 −1.91 * * F 1.24 2.63 Glu 157 A A . . . . .2.18 −1.49 * * F 1.58 1.50 Glu 158 A A . . . . . 2.13 −0.73 * * F 1.921.20 Asn 159 . . . . T T . 1.82 −0.33 * * F 2.76 2.76 Gly 160 . . . . TT . 1.47 −0.53 * * F 3.40 2.30 Tyr 161 . . . . T T . 1.77 0.23 . . F2.16 2.08 Asn 162 . . . . . T C 1.47 0.73 . . F 1.32 1.31 Thr 163 . . .. . . C 0.77 0.71 . . . 0.63 1.77 Tyr 164 . . B . . . . 0.77 1.07 . * .−0.06 0.98 Ala 165 . . B . . . . 0.82 0.71 . * . −0.40 0.98 Ser 166 . .B . . T . 1.07 1.23 . * . −0.20 0.71 Phe 167 . . B . . T . 1.03 1.14 . *. −0.20 0.79 Asn 168 . . . . T T . 1.34 0.89 . * . 0.35 1.06 Trp 169 . .. . T T . 1.24 0.79 . * . 0.35 1.27 Gln 170 . . . . . . C 1.94 0.83 * *. 0.11 1.45 His 171 . . . . . T C 2.24 0.04 * * . 0.77 1.77 Asn 172 . .. . . T C 2.34 0.04 * * F 1.08 2.92 Gly 173 . . . . T T . 2.10 −0.26 * *F 2.04 1.67 Arg 174 . . . . T T . 1.53 0.10 * * F 1.60 1.92 Gln 175 . .B B . . . 0.94 0.24 * . . 0.34 0.89 Met 176 . . B B . . . 0.17 0.34 * .. 0.18 0.90 Tyr 177 . . B B . . . 0.17 0.60 * * . −0.28 0.38 Val 178 . .B B . . . 0.17 1.00 . * . −0.44 0.35 Ala 179 . . B B . . . 0.10 1.03 . *. −0.60 0.35 Leu 180 . . B B . . . −0.24 0.41 . * . −0.30 0.45 Asn 181 .. . . T T . −0.23 0.09 . * F 1.25 0.60 Gly 182 . . . . T T . −0.20−0.06 * * F 2.15 0.60 Lys 183 . . . . T T . 0.77 −0.13 * * F 2.60 1.13Gly 184 . . . . . T C 1.47 −0.81 * * F 3.00 1.37 Ala 185 . . . . . . C1.93 −1.21 * * F 2.50 2.72 Pro 186 . . B . . T . 1.93 −1.21 * . F 2.201.35 Arg 187 . . B . . T . 2.32 −0.81 * . F 1.90 2.35 Arg 188 . . B . .T . 1.97 −1.24 * . F 1.60 4.66 Gly 189 . . B . . T . 2.42 −1.26 * . F1.30 4.35 Gln 190 . . B . . . . 3.12 −1.69 * . F 1.10 4.35 Lys 191 . . B. . . . 3.38 −1.69 * . F 1.10 4.35 Thr 192 . . B . . . . 3.27 −1.69 * .F 1.44 8.79 Arg 193 . . B . . . . 2.84 −1.71 . . F 1.78 8.16 Arg 194 . .. . T . . 2.89 −1.63 * . F 2.52 5.89 Lys 195 . . . . T . . 2.30 −1.24 *. F 2.86 5.47 Asn 196 . . . . T T . 2.22 −1.23 . * F 3.40 2.82 Thr 197 .. . . . T C 1.83 −0.73 . . F 2.86 1.96 Ser 198 . . . . . T C 0.91 0.06 .. F 1.47 0.85 Ala 199 . . B . . T . 0.59 0.74 . . . 0.48 0.44 His 200 .. B . . . . −0.06 0.77 . . . −0.06 0.47 Phe 201 . . B B . . . −0.910.90 * . . −0.60 0.34 Leu 202 . . B B . . . −1.46 1.16 . . . −0.60 0.25Pro 203 . . B B . . . −1.19 1.30 . . . −0.60 0.14 Met 204 . . B B . . .−0.90 1.30 * . . −0.60 0.22 Val 205 A . . B . . . −1.26 0.90 * . . −0.600.35 Val 206 A . . B . . . −0.94 0.64 . . . −0.60 0.29 His 207 A . . B .. . −0.52 0.64 . . . −0.60 0.38 Ser 208 A . . B . . . −0.70 0.46 . . .−0.60 0.65

Among highly preferred fragments in this regard are those that compriseregions of KGF-2 that combine several structural features, such asseveral of the features set out above.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of KGF-2 therebyeffectively generating agonists and antagonists of KGF-2. See generally,U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and5,837,458; and Patten, P. A. et al., Curr. Opinion Biotechnol. 8:724–33(1997); Harayama, S., Trends Biotechnol. 16(2):76–82 (1998); Hansson, L.O. et al., J. Mol. Biol. 287:265–76 (1999); and Lorenzo, M. M. andBlasco, R., Biotechniques 24(2):308–13 (1998) (each of these patents andpublications are hereby incorporated by reference).

In one embodiment, alteration of KGF-2 polynucleotides and correspondingpolypeptides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments into a desired KGF-2 moleculeby homologous, or site-specific, recombination. In another embodiment,KGF-2 polynucleotides and corresponding polypeptides may be altered bybeing subjected to random mutagenesis by error-prone PCR, randomnucleotide insertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of KGF-2 may be recombined with one or more components,motifs, sections, parts, domains, fragments, etc. of one or moreheterologous molecules. In preferred embodiments, the heterologousmolecules are KGF-2 family members. In further preferred embodiments,the heterologous molecule is a growth factor such as, for example,platelet-derived growth factor (PDGF), insulin-like growth factor(IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor(EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogeneticprotein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B,decapentaplegic (dpp), 60A, OP-2, dorsalin, growth differentiationfactors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2,TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).Other preferred fragments are biologically active KGF-2 fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the KGF-2 polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, the polypeptide of the present invention, thecompound to be screened and 3[H] thymidine under cell culture conditionswhere the fibroblast cell would normally proliferate. A control assaymay be performed in the absence of the compound to be screened andcompared to the amount of fibroblast proliferation in the presence ofthe compound to determine if the compound stimulates proliferation bydetermining the uptake of 3[H] thymidine in each case. The amount offibroblast cell proliferation is measured by liquid scintillationchromatography which measures the incorporation of 3[H] thymidine. Bothagonist and antagonist compounds may be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the KGF-2 receptor is measured and the ability of thecompound to bind to the receptor and elicit a second messenger responseis measured to determine if the compound is a potential agonist orantagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting the KGF-2 molecule.Moreover, the assays can discover agents which may inhibit or enhancethe production of KGF-2 from suitably manipulated cells or tissues.

Therefore, the invention includes a method of identifying compoundswhich bind to KGF-2 comprising the steps of: (a) incubating a candidatebinding compound with KGF-2; and (b) determining if binding hasoccurred. Moreover, the invention includes a method of identifyingagonists/antagonists comprising the steps of: (a) incubating a candidatecompound with KGF-2, (b) assaying a biological activity, and (c)determining if a biological activity of KGF-2 has been altered.

Also, one could identify molecules bind KGF-2 experimentally by usingthe beta-pleated sheet regions disclosed in FIG. 4 and Table 1.Accordingly, specific embodiments of the invention are directed topolynucleotides encoding polypeptides which comprise, or alternativelyconsist of, the amino acid sequence of each beta pleated sheet regionsdisclosed in FIG. 3/Table 1.

Additional embodiments of the invention are directed to polynucleotidesencoding KGF-2 polypeptides which comprise, or alternatively consist of,any combination or all of the beta pleated sheet regions disclosed inFIG. 4/Table 1. Additional preferred embodiments of the invention aredirected to polypeptides which comprise, or alternatively consist of,the KGF-2 amino acid sequence of each of the beta pleated sheet regionsdisclosed in FIG. 4/Table 1. Additional embodiments of the invention aredirected to KGF-2 polypeptides which comprise, or alternatively consistof, any combination or all of the beta pleated sheet regions disclosedin FIG. 4/Table 1.

Other preferred embodiments of the invention are fragments of KGF-2which bind to the KGF-2 receptor. Fragments which bind to the KGF-2receptor may be useful as agonists or antagonists of KGF-2. For example,fragments of KGF-2 which bind the receptor may prevent binding to KGF-2and active portions thereof. Other fragments may bind to the receptorand specifically deactivate the receptor and receptor activation or mayspecifically antibodies that recognize the receptor-ligand complex, and,preferably, do not specifically recognize the unbound receptor or theunbound ligand. Likewise, included in the invention are fragments whichactivate the receptor. These fragments may act as receptor agonists,i.e., potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The fragments may be specified asagonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein.

Non-limiting examples of fragments of KGF-2 which bind the KGF-2receptor include amino acids 147–155, 95–105, 78–94, 119–146, 70–94,78–105, 114–146, 70–105, 86–124, 100–139, 106–146, 160–209, and/or156–209 of SEQ ID NO:2. Also preferred are polynucleotides encoding suchpolypeptides.

Other preferred fragments are biologically active KGF-2 fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the KGF-2 polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

However, many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 and 613 of SEQID NO:1, b is an integer of 15 to 627, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:1, and where bis greater than or equal to a +14.

Amino Terminal and Carboxy Terminal Deletions

Various members of the FGF family have been modified using recombinantDNA technology. Positively charged molecules have been substituted ordeleted in both aFGF and bFGF that are important for heparin binding.The modified molecules resulted in reduced heparin binding activity.Accordingly, it is known that the amount of modified moleculesequestered by heparin in a patient would be reduced, increasing thepotency as more FGF would reach the appropriate receptor. (EP 0 298723).

Native KGF-2 is relatively unstable in the aqueous state and itundergoes chemical and physical degradation resulting in loss ofbiological activity during processing and storage. Native KGF-2 is alsoprone to aggregation in aqueous solution, at elevated temperatures andit becomes inactivated under acidic conditions.

In order to improve or alter one or more characteristics of nativeKGF-2, protein engineering may be employed. Ron et al., J. Biol. Chem.,268(4): 2984–2988 (1993) reported modified KGF proteins that had heparinbinding activity even if the 3, 8, or 27 amino terminal amino acidresidues were missing. The deletion of 3 and 8 amino acids had fullactivity. More deletions of KGF have been described in PCT/IB95/00971.The deletion of carboxyterminal amino acids can enhance the activity ofproteins. One example is interferon gamma that shows up to ten timeshigher activity by deleting ten amino acid residues from the carboxyterminus of the protein (Döbeli et al., J. of Biotechnology 7:199–216(1988)). Thus, one aspect of the invention is to provide polypeptideanalogs of KGF-2 and nucleotide sequences encoding such analogs thatexhibit enhanced stability (e.g., when exposed to typical pH, thermalconditions or other storage conditions) relative to the native KGF-2polypeptide.

Particularly preferred KGF-2 polypeptides are shown below (numberingstarts with the first amino acid in the protein (Met) (FIGS. 1A–1C (SEQID NO:2)):

Thr (residue 36) -- Arg (65) -- Ser (208) Ser (residue 208) Cys (37)--Ser (208) Val (67) -- Ser (208) Gln (38) -- Ser (208) Ser (69) -- Ser(208) Ala (39) -- Ser (208) Val (77) -- Ser (208) Leu (40) -- Ser (208)Arg (80) -- Ser (208) Gly (41) -- Ser (208) Met(1), Thr (36), or Cys(37) -- His (207) Gln (42) -- Ser (208) Met (1), Thr (36), or Cys (37)-- Val (206) Asp (43) -- Ser (208) Met (1), Thr (36), or Cys (37) -- Val(205) Met (44) -- Ser (208) Met(1), Thr (36), or Cys (37) -- Met (204)Val (45) -- Ser (208) Met(1), Thr (36), or Cys (37) -- Pro (203) Ser(46) -- Ser (208) Met(1), Thr (36), or Cys(37) -- Leu (202) Pro (47) --Ser (208) Met(1), Thr (36), or Cys (37) -- Phe (201) Glu (48) -- Ser(208) Met(1), Thr (36), or Cys (37) -- His (200) Ala (49) -- (Ser (208)Met(1), Thr (36), or Cys (37) -- Ala (199) Thr (50) -- Ser (208) Met(1), Thr (36), or Cys (37) -- Ser (198) Asn (51) -- Ser (208) Met (1),Thr (36), or Cys (37) -- Thr (197) Ser (52) -- Ser (208) Met(1), Thr(36), or Cys (37) -- Asn (196) Ser (53) -- Ser (208) Met(1), Thr (36),or Cys (37) -- Lys (195) Ser (54) -- Ser (208) Met (1), Thr (36), or Cys(37) -- Arg (194) Ser (55) -- Ser (208) Met(1), Thr (36), or Cys (37) --Arg (193) Ser (56) -- Ser (208) Met(1), Thr (36), or Cys (37) -- Thr(192) Phe (57) -- Ser (208) Met(1), Thr (36), or Cys (37) -- Lys (191)Ser (59) -- Ser (208) Met(1), Thr (36), or Cys (37) -- Arg (188) Ser(62) -- Ser (208) Met(1), Thr (36), or Cys (37) -- Arg (187) Ala (63) --Ser (208) Met(1), Thr (36), or Cys (37) -- Lys (183) Gly (64) -- Ser(208)

Preferred embodiments include the N-terminal deletions Ala (63)—Ser(208) (KGF 2Δ28) (SEQ ID NO:68) and Ser (69)—Ser (208) (KGF 2Δ33) (SEQID NO:96). Other preferred N-terminal and C-terminal deletion mutantsare described in Examples 13 and 16 (c) of the specification andinclude: Ala (39)—Ser (208) (SEQ ID NO:116); Pro (47)—Ser (208) of FIGS.1A–1C (SEQ ID NO:2); Val (77)—Ser (208) (SEQ ID NO:70); Glu (93)—Ser(208) (SEQ ID NO:72); Glu (104)—Ser (208) (SEQ ID NO:74); Val (123)—Ser(208) (SEQ ID NO:76); and Gly (138)—Ser (208) (SEQ ID NO:78). Otherpreferred C-terminal deletion mutants include: Met (1), Thr (36), or Cys(37)—Lys (153) of FIGS. 1A–1C (SEQ ID NO:2).

Also included by the present invention are deletion mutants having aminoacids deleted from both the—terminus and the C-terminus. Such mutantsinclude all combinations of the N-terminal deletion mutants andC-terminal deletion mutants described above, e.g., Ala (39) His (200) ofFIGS. 1A–1C (SEQ ID NO:2), Met (44)—Arg (193) of FIG. 1 (SEQ ID NO:2),Ala (63)—Lys (153) of FIGS. 1A–1C (SEQ ID NO:2), Ser (69)—Lys (153) ofFIGS. 1A–1C (SEQ ID NO:2), etc. etc. etc. . . . . Those combinations canbe made using recombinant techniques known to those skilled in the art.

Thus, in one aspect, N-terminal deletion mutants are provided by thepresent invention. Such mutants include those comprising the amino acidsequence shown in FIGS. 1A–1C (SEQ ID NO:2) except for a deletion of atleast the first 38 N-terminal amino acid residues (i.e., a deletion ofat least Met (1)—Gln (38)) but not more than the first 147 N-terminalamino acid residues of FIGS. 1A–1C (SEQ ID NO:2). Alternatively, thedeletion will include at least the first 38 N-terminal amino acidresidues (i.e., a deletion of at least Met (1)—Gln (38)) but not morethan the first 137 N-terminal amino acid residues of FIGS. 1A–1C (SEQ IDNO:2). Alternatively, the deletion will include at least the first 46N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2).Alternatively, the deletion will include at least the first 62N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2).Alternatively, the deletion will include at least the first 68N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2).Alternatively, the deletion will include at least the first 76N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2).Alternatively, the deletion will include at least the first 92N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2).Alternatively, the deletion will include at least the first 103N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2).Alternatively, the deletion will include at least the first 122N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2).

In addition to the ranges of N-terminal deletion mutants describedabove, the present invention is also directed to all combinations of theabove described ranges, e.g., deletions of at least the first 62N-terminal amino acid residues but not more than the first 68 N-terminalamino acid residues of FIGS. 1A–1C (SEQ ID NO:2); deletions of at leastthe first 62 N-terminal amino acid residues but not more than the first76 N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2);deletions of at least the first 62 N-terminal amino acid residues butnot more than the first 92 N-terminal amino acid residues of FIGS. 1A–1C(SEQ ID NO:2); deletions of at least the first 62 N-terminal amino acidresidues but not more than the first 103 N-terminal amino acid residuesof FIGS. 1A–1C (SEQ ID NO:2); deletions of at least the first 68N-terminal amino acid residues but not more than the first 76 N-terminalamino acid residues of FIGS. 1A–1C (SEQ ID NO:2); deletions of at leastthe first 68 N-terminal amino acid residues but not more than the first92 N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2);deletions of at least the first 68 N-terminal amino acid residues butnot more than the first 103 N-terminal amino acid residues of FIGS.1A–1C (SEQ ID NO:2); deletions of at least the first 46 N-terminal aminoacid residues but not more than the first 62 N-terminal amino acidresidues of FIGS. 1A–1C (SEQ ID NO:2); deletions of at least the first46 N-terminal amino acid residues but not more than the first 68N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2); deletionsof at least the first 46 N-terminal amino acid residues but not morethan the first 76 N-terminal amino acid residues of FIGS. 1A–1C (SEQ IDNO:2); etc. etc. etc. . . .

In another aspect, C-terminal deletion mutants are provided by thepresent invention. Preferably, the N-terminal amino acid residue of saidC-terminal deletion mutants is amino acid residue 1 (Met), 36 (Thr), or37 (Cys) of FIGS. 1A–1C (SEQ ID NO:2). Such mutants include thosecomprising the amino acid sequence shown in FIGS. 1A–1C (SEQ ID NO:2)except for a deletion of at least the last C-terminal amino acid residue(Ser (208)) but not more than the last 55 C-terminal amino acid residues(i.e., a deletion of amino acid residues Glu (154)—Ser (208)) of FIGS.1A–1C (SEQ ID NO:2). Alternatively, the deletion will include at leastthe last C-terminal amino acid residue but not more than the last 65C-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2).Alternatively, the deletion will include at least the last 10 C-terminalamino acid residues but not more than the last 55 C-terminal amino acidresidues of FIGS. 1A–1C (SEQ ID NO:2).

Alternatively, the deletion will include at least the last 20 C-terminalamino acid residues but not more than the last 55 C-terminal amino acidresidues of FIGS. 1A–1C (SEQ ID NO:2). Alternatively, the deletion willinclude at least the last 30 C-terminal amino acid residues but not morethan the last 55 C-terminal amino acid residues of FIGS. 1A–1C (SEQ IDNO:2). Alternatively, the deletion will include at least the last 40C-terminal amino acid residues but not more than the last 55 C-terminalamino acid residues of FIGS. 1A–1C (SEQ ID NO:2). Alternatively, thedeletion will include at least the last 50 C-terminal amino acidresidues but not more than the last 55 C-terminal amino acid residues ofFIGS. 1A–1C (SEQ ID NO:2).

In addition to the ranges of C-terminal deletion mutants describedabove, the present invention is also directed to all combinations of theabove described ranges, e.g., deletions of at least the last C-terminalamino acid residue but not more than the last 10 C-terminal amino acidresidues of FIGS. 1A–1C (SEQ ID NO:2); deletions of at least the lastC-terminal amino acid residue but not more than the last 20 C-terminalamino acid residues of FIGS. 1A–1C (SEQ ID NO:2); deletions of at leastthe last C-terminal amino acid residue but not more than the last 30C-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2); deletionsof at least the last C-terminal amino acid residue but not more than thelast 40 C-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2);deletions of at least the last 10 C-terminal amino acid residues but notmore than the last 20 C-terminal amino acid residues of FIGS. 1A–1C (SEQID NO:2); deletions of at least the last 10 C-terminal amino acidresidues but not more than the last 30 C-terminal amino acid residues ofFIGS. 1A–1C (SEQ ID NO:2); deletions of at least the last 10 C-terminalamino acid residues but not more than the last 40 C-terminal amino acidresidues of FIGS. 1A–1C (SEQ ID NO:2); deletions of at least the last 20C-terminal amino acid residues but not more than the last 30 C-terminalamino acid residues of FIGS. 1A–1C (SEQ ID NO:2); etc. etc. etc. . . .

In yet another aspect, also included by the present invention aredeletion mutants having amino acids deleted from both the — terminal andC-terminal residues. Such mutants include all combinations of theN-terminal deletion mutants and C-terminal deletion mutants describedabove. Such mutants include those comprising the amino acid sequenceshown in FIGS. 1A–1C (SEQ ID NO:2) except for a deletion of at least thefirst 46 N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2) and adeletion of at least the last C-terminal amino acid residue but not morethan the last 55 C-terminal amino acid residues of FIGS. 1A–1C (SEQ IDNO:2). Alternatively, a deletion can include at least the first 62, 68,76, 92, 103, or 122 N-terminal amino acids but not more than the first137 N-terminal amino acid residues of FIGS. 1A–1C (SEQ ID NO:2) and adeletion of at least the last 10, 20, 30, 40, or 50 C-terminal aminoacid residues but not more than the last 55 C-terminal amino acidresidues of FIGS. 1A–1C (SEQ ID NO:2). Further included are allcombinations of the above described ranges.

Substitution of Amino Acids

A further aspect of the present invention also includes the substitutionof amino acids. Native mature KGF-2 contains 44 charged residues, 32 ofwhich carry a positive charge. Depending on the location of suchresidues in the protein's three dimensional structure, substitution ofone or more of these clustered residues with amino acids carrying anegative charge or a neutral charge may alter the electrostaticinteractions of adjacent residues and may be useful to achieve increasedstability and reduced aggregation of the protein. Aggregation ofproteins cannot only result in a loss of activity but be problematicwhen preparing pharmaceutical formulations, because they can beimmunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331–340 (1967),Robbins et al., Diabetes 36: 838–845 (1987), Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10: 307–377 (1993)). Any modificationshould give consideration to minimizing charge repulsion in the tertiarystructure of the protein molecule. Thus, of special interest aresubstitutions of charged amino acid with another charge and with neutralor negatively charged amino acids. The latter results in proteins with areduced positive charge to improve the characteristics of KGF-2. Suchimprovements include increased stability and reduced aggregation of theanalog as compared to the native KGF-2 protein.

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361: 266–268(1993), described certain TNF alpha mutations resulting in selectivebinding of TNF alpha to only one of the two known TNF receptors.

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of a KGF-2 polypeptide having an aminoacid sequence which contains at least one amino acid substitution, butnot more than 50 amino acid substitutions, even more preferably, notmore than 40 amino acid substitutions, still more preferably, not morethan 30 amino acid substitutions, and still even more preferably, notmore than 20 amino acid substitutions. Of course, in order ofever-increasing preference, it is highly preferable for a peptide orpolypeptide to have an amino acid sequence which comprises the aminoacid sequence of a KGF-2 polypeptide, which contains at least one, butnot more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5,4, 3, 2 or 1 amino acid substitutions. In specific embodiments, thenumber of additions, substitutions, and/or deletions in the amino acidsequence of FIGS. 1A–1C or fragments thereof (e.g., the mature formand/or other fragments described herein), is 1–5,5–10, 5–25, 5–50, 10–50or 50–150, conservative amino acid substitutions are preferable.

KGF-2 molecules may include one or more amino acid substitutions,deletions or additions, either from natural mutation or humanmanipulation. The mutations can be made in full-length KGF-2, matureKGF-2, any other appropriate fragments of KGF-2, for example, A63-S208,S69-S208, V77-S208, R80-S208 or E93-S208. Examples of some preferredmutations are: Ala (49) Gln, Asn (51) Ala, Ser (54) Val, Ala (63) Pro,Gly (64) Glu, Val (67) Thr, Trp (79) Val, Arg (80) Lys, Lys (87) Arg,Tyr (88) Trp, Phe (89) Tyr, Lys (91) Arg, Ser (99) Lys, Lys (102) Gln,Lys 103(Glu), Glu (104) Met, Asn (105) Lys, Pro (107) Asn, Ser (109)Asn, Leu (111) Met, Thr (114) Arg, Glu(117) Ala, Val (120) Ile, Val(123) Ile, Ala (125) Gly, Ile (126) Val, Asn (127) Glu, Asn (127) Gln,Tyr (130) Phe, Met (134) Thr, Lys (136) Glu, Lys (137) Glu, Gly (142)Ala, Ser (143) Lys, Phe (146) Ser, Asn (148) Glu, Lys (151) Asn, Leu(152) Phe, Glu (154) Gly, Glu (154) Asp, Arg (155) Leu, Glu (157) Leu,Gly (160) His, Phe (167) Ala, Asn (168) Lys, Gln (170) Thr, Arg (174)Gly, Tyr (177) Phe, Gly (182) Gln, Ala (185) Val, Ala (185) Leu, Ala(185) Ile, Arg (187) Gln (190) Lys, Lys (195) Glu, Thr (197) Lys, Ser(198) Thr, Arg (194) Glu, Arg (194) Gln, Lys (191) Glu, Lys (191) Gln,Arg (188) Glu, Arg (188) Gln, Lys (183) Glu, Arg (187) Ala, Arg (188)Ala, Arg 174 (Ala), Lys (183) Ala, Lys (144) Ala, Lys (151) Ala, Lys(153) Ala, Lys (136) Ala, Lys (137) Ala, and Lys (139) Ala.

By the designation, for example, Ala (49) Gln is intended that the Alaat position 49 of FIGS. 1A–1C (SEQ ID NO:2) is replaced by Gln.

Additionally, the following mutants are particularly preferred: S69-S208with a point mutation at R188E; S69-S208 with a point mutation at K191E;S69-S208, with a point mutation at K149E; S69-S208 with a point mutationat K183Q; S69-S208 with a point mutation at K183E; A63-S208 with a pointmutation at R68G; A63-S208 with a point mutation at R68S; A63-S208 witha point mutation at R68A; A63-S208 with point mutations at R78A, R80Aand K81A; A63-S208 with point mutations at K81A, K87A and K91A; A63-S208with point mutations at R78A, R80A, K81A, K87A and K91A; A63-S208 withpoint mutations at K136A, K137A, K139A and K144A; A63-S208 with pointmutations at K151A, K153A and K155A; A63-S208 with point mutations atR68G, R78A, R80A, and K81A; A63-S208 with point mutations at R68G, K81A,K87A and K91A; A63-S208 with point mutations at R68G, R78A, R80A, K81A,K87A and K91A; A63-S208 with point mutations at R68G, K136A, K137A,K139A, and K144A; A63-208 with point mutations at R68G, K151A, K153A,and R155A; A63-S208 with point mutations at R68S, R78A, R80A, and K81A;A63-S208 with point mutations at R68S, K81A, R87A and K91A; A63-S208with point mutations at R68S, R78A, R80A, K81A, K87A and K91A; A63-S208with point mutations at R68S, K136A, K137A, K139A, and K144A; A63-208with point mutations at R68S, K151A, K153A, and R155A; A63-S208 withpoint mutations at R68A, R78A, R80A and K81A; A63-S208 with pointmutations at R68A, K81A, K87A, and K91A; A63-S208 with point mutationsat R68A, R78A, R80A, K81A, K87A, and K91A; A63-S208 with point mutationsat R68A, K136A, K137A, K139A and K144A; and A63-S208 with pointmutations at R68A, K151A, K153A and R155A. Also preferred are: A63-S208with the positively charged residues between and including R68 to K91are replaced with alanine [A63-S208 (R68-K91A)]; full length KGF-2 withthe positively charged residues between and including R68 to K91replaced with alanine [KGF-2(R68-K91A)]; A63-S208 with the positivelycharged residues between and including R68 to K91 replaced with neutralresidues, such as G, S and/or A; full length KGF-2 with the positivelycharged residues between and including R68 to K91 replaced with neutralresidues, such as G, S and/or A; A63-S208 with the positively chargedresidues between and including R68 to K91 replaced with negativelycharged acidic residues, such as D and/or E; full length KGF-2 with thepositively charged residues between and including R68 to K91 replacedwith negatively charged acidic residues, such as D and/or E; full lengthKGF-2 with point mutations at R78A, R80A, and K81A; full length KGF-2with point mutations at K81A, K87A and K91A; full length KGF-2 with apoint mutation at R68G; full length KGF-2 with a point mutation at R68S;full length KGF-2 with a point mutation at R68A; A63-S208 with pointmutations at R174A and K183A; and A63-S208 with point mutations at R187Aand R188A.

Also preferred is A63-S208 with a point mutation at R188E, K191E, K149E,K183Q, or K183E; S69-S208 with point mutations at R78A, R80A and K81A;S69-S208 with point mutations at K81A, K87A and K91A; S69-S208 withpoint mutations at R174A and K183A; S69-S208 with point mutations atR187A and R188A; V77-S208 with a point mutation at R188E, K191E, K149E,K183Q, or K183E; V77-S208 with point mutations at R78A, R80A and K81A;V77-S208 with point mutations at K81A, K87A and K91A; V77-S208 withpoint mutations at R174A and K183A; V77-S208 with point mutations atR187A and R188A; R80-S208 with a point mutation at R188E, K191E, K149E,K183Q, or K183E; R80-S208 with point mutations at R174A and K183A;R80-S208 with point mutations at R187A and R188A; E93-S208 with a pointmutation at R188E, K191E, K149E, K183Q, or K183E; E93-S208 with pointmutations at R174A and K183A; or E93-S208 with point mutations at R187Aand R188A.

All of the above point mutations may also be made in the full lengthKGF-2, the mature KGF-2, or any other fragment of KGF-2 describedherein. By the designation, for sample, R188E is intended that theArginine at position 188 is replaced with a Glutamic Acid.

In addition site directed mutations may be made at each amino acids ofKGF-2, preferably between amino acids A63 to E93. Each amino acid can bereplaced by any of the other 19 remaining amino acids. For examplepreferred mutations include: A63 replaced with C, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, W, or Y; G64 replaced with A, C, D, E, F, H,I, K, L, M, N, P, Q, R, S, T, V, W, or Y; R65 replaced with A, C, D, E,F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; H66 replaced with A, C,D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; V67 replaced withA, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; R68 replacedwith A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; S69replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, orY; Y70 replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, or W; N71 replaced with A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,T, V, W, or Y; H72 replaced with A, C, D, E, F, G, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; L73 replaced with A, C, D, E, F, G, H, I, K, M, N,P, Q, R, S, T, V, W, or Y; Q74 replaced with A, C, D, E, F, G, H, I, K,L, M, N, P, R, S, T, V, W, or Y; G75 replaced with A, C, D, E, F, H, I,K, L, M, N, P, Q, R, S, T, V, W, or Y; D76 replaced with A, C, E, F, G,H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; V77 replaced with A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; R78 replaced with A,C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; W79 replacedwith A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; R80replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, orY; K81 replaced with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V,W, or Y; L82 replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S,T, V, W, or Y; F83 replaced with A, C, D, E, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y; S84 replaced with A, C, D, E, F, G, H, I, K, L, M,N, P, Q, R, T, V, W, or Y; F85 replaced with A, C, D, E, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y; T86 replaced with A, C, D, E, F, G, H,I, K, L, M, N, P, Q, R, S, V, W, or Y; K87 replaced with A, C, D, E, F,G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; Y88 replaced with A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; F89 replaced with A,C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; L90 replacedwith A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; K91replaced with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, orY; 192 replaced with A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V,W, or Y; and/or E93 replaced with A, C, D, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y.

These mutations can be made in the N-terminal deletion constructspreviously described, particularly constructs beginning with amino acidsM1, T36, C37, or A63. Additionally, more than one amino acid (e.g. 2, 3,4, 5, 6, 7, 8, 9 and 10) can be replaced in this region (A63 to E93)with other amino acids. The resulting constructs can be screened forloss of heparin binding, loss of KGF-2 activity, and/or loss ofenzymatic cleavage between amino acids R68 and S69.

Preferred mutations are located at amino acid positions R68 and S69 inN-terminal deletion constructs M1, T36, C37 and A63, as well asmutations in the heparin binding domain, of all of the above listedN-terminal mutants, especially T36, C37, A63, S69, V77, R80 or E93. Theheparin binding domain is between Arg174 and Lys 183. Preferred Arg68mutants replace the arginine with Gly, Ser or Ala; preferred Arg187mutants replace the arginine with alanine.

Two ways in which mutations can be made is either by site directedmutagenesis or accelerated mutagenesis (Kuchner and Arnold, Tibtech5:523–530 (1997); Crameri et al., Nature (1998); and Christians et al.,Nature Biotechnology 17:259264 (1999)). These methods are well known inthe art.

Changes are preferably of minor nature, such as conservative amino acidsubstitutions that do not significantly affect the folding or activityof the protein. Examples of conservative amino acid substitutions knownto those skilled in the art are set forth below:

-   Aromatic:    -   phenylalanine    -   tryptophan    -   tyrosine-   Hydrophobic:    -   leucine    -   isoleucine    -   valine-   Polar:    -   glutamine    -   asparagine-   Basic:    -   arginine    -   lysine    -   histidine-   Acidic:    -   aspartic acid    -   glutamic acid-   Small:    -   alanine    -   serine    -   threonine    -   methionine    -   glycine

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of substitutions for any given KGF-2polypeptide will not be more than 50, 40, 30, 20, 10, 5, or 3, dependingon the objective. For example, a number of substitutions that can bemade in the C-terminus of KGF-2 to improve stability are described aboveand in Example 22.

Particularly preferred are KGF-2 molecules with conservative amino acidsubstitutions, including: M1 replaced with A, G, I, L, S, T, or V; W2replaced with F, or Y; K3 replaced with H, or R; W4 replaced with F, orY; 15 replaced with A, G, L, S, T, M, or V; L6 replaced with A, G, I, S,T, M, or V; T7 replaced with A, G, I, L, S, M, or V; H8 replaced with K,or R; A10 replaced with G, I, L, S, T, M, or V; S11 replaced with A, G,I, L, T, M, or V; A12 replaced with G, I, L, S, T, M, or V; F13 replacedwith W, or Y; H15 replaced with K, or R; L16 replaced with A, G, I, S,T, M, or V; G18 replaced with A, I, L, S, T, M, or V; F24 replaced withW, or Y; L25 replaced with A, G, I, S, T, M, or V; L26 replaced with A,G, I, S, T, M, or V; L27 replaced with A, G, I, S, T, M, or V; F28replaced with W, or Y; L29 replaced with A, G, I, S, T, M, or V; V30replaced with A, G, I, L, S, T, or M; S31 replaced with A, G, I, L, T,M, or V; S32 replaced with A, G, I, L, T, M, or V; V33 replaced with A,G, I, L, S, T, or M; V35 replaced with A, G, I, L, S, T, or M; T36replaced with A, G, I, L, S, M, or V; Q38 replaced with N; A39 replacedwith G, I, L, S, T, M, or V; L40 replaced with A, G, I, S, T, M, or V;G41 replaced with A, I, L, S, T, M, or V; Q42 replaced with N; D43replaced with E; M44 replaced with A, G, I, L, S, T, or V; V45 replacedwith A, G, I, L, S, T, or M; S46 replaced with A, G, I, L, T, M, or V;E48 replaced with D; A49 replaced with G, I, L, S, T, M, or V; T50replaced with A, G, I, L, S, M, or V; N51 replaced with Q; S52 replacedwith A, G, I, L, T, M, or V; S53 replaced with A, G, I, L, T, M, or V;S54 replaced with A, G, I, L, T, M, or V; S55 replaced with A, G, I, L,T, M, or V; S56 replaced with A, G, I, L, T, M, or V; F57 replaced withW, or Y; S58 replaced with A, G, I, L, T, M, or V; S59 replaced with A,G, I, L, T, M, or V; S61 replaced with A, G, I, L, T, M, or V; S62replaced with A, G, I, L, T, M, or V; A63 replaced with G, I, L, S, T,M, or V; G64 replaced with A, I, L, S, T, M, or V; R65 replaced with H,or K; H66 replaced with K, or R; V67 replaced with A, G, I, L, S, T, orM; R68 replaced with H, or K; S69 replaced with A, G, I, L, T, M, or V;Y70 replaced with F, or W; N71 replaced with Q; H72 replaced with K, orR; L73 replaced with A, G, I, S, T, M, or V; Q74 replaced with N; G75replaced with A, I, L, S, T, M, or V; D76 replaced with E; V77 replacedwith A, G, I, L, S, T, or M; R78 replaced with H, or K; W79 replacedwith F, or Y; R80 replaced with H, or K; K81 replaced with H, or R; L82replaced with A, G, I, S, T, M, or V; F83 replaced with W, or Y; S84replaced with A, G, I, L, T, M, or V; F85 replaced with W, or Y; T86replaced with A, G, I, L, S, M, or V; K87 replaced with H, or R; Y88replaced with F, or W; F89 replaced with W, or Y; L90 replaced with A,G, I, S, T, M, or V; K91 replaced with H, or R; 192 replaced with A, G,L, S, T, M, or V; E93 replaced with D; K94 replaced with H, or R; N95replaced with Q; G96 replaced with A, I, L, S, T, M, or V; K97 replacedwith H, or R; V98 replaced with A, G, I, L, S, T, or M; S99 replacedwith A, G, I, L, T, M, or V; G100 replaced with A, I, L, S, T, M, or V;T101 replaced with A, G, I, L, S, M, or V; K102 replaced with H, or R;K103 replaced with H, or R; E104 replaced with D; N105 replaced with Q;Y108 replaced with F, or W; S109 replaced with A, G, I, L, T, M, or V;I110 replaced with A, G, L, S, T, M, or V; L111 replaced with A, G, I,S, T, M, or V; E 112 replaced with D; I113 replaced with A, G, L, S, T,M, or V; T114 replaced with A, G, I, L, S, M, or V; S115 replaced withA, G, I, L, T, M, or V; V116 replaced with A, G, I, L, S, T, or M; E117replaced with D; I118 replaced with A, G, L, S, T, M, or V; G119replaced with A, I, L, S, T, M, or V; V120 replaced with A, G, I, L, S,T, or M; V121 replaced with A, G, I, L, S, T, or M; A122 replaced withG, I, L, S, T, M, or V; V123 replaced with A, G, I, L, S, T, or M; K124replaced with H, or R; A125 replaced with G, I, L, S, T, M, or V; I126replaced with A, G, L, S, T, M, or V; N127 replaced with Q; S128replaced with A, G, I, L, T, M, or V; N129 replaced with Q; Y130replaced with F, or W; Y131 replaced with F, or W; L132 replaced with A,G, I, S, T, M, or V; A133 replaced with G, I, L, S, T, M, or V; M134replaced with A, G, I, L, S, T, or V; N135 replaced with Q; K136replaced with H, or R; K137 replaced with H, or R; G138 replaced with A,I, L, S, T, M, or V; K139 replaced with H, or R; L140 replaced with A,G, I, S, T, M, or V; Y141 replaced with F, or W; G142 replaced with A,I, L, S, T, M, or V; S143 replaced with A, G, I, L, T, M, or V; K144replaced with H, or R; E145 replaced with D; F146 replaced with W, or Y;N147 replaced with Q; N148 replaced with Q; D149 replaced with E; K151replaced with H, or R; L152 replaced with A, G, I, S, T, M, or V; K153replaced with H, or R; E154 replaced with D; R155 replaced with H, or K;I156 replaced with A, G, L, S, T, M, or V; E157 replaced with D; E158replaced with D; N159 replaced with Q; G160 replaced with A, I, L, S, T,M, or V; Y161 replaced with F, or W; N162 replaced with Q; T163 replacedwith A, G, I, L, S, M, or V; Y164 replaced with F, or W; A165 replacedwith G, I, L, S, T, M, or V; S166 replaced with A, G, I, L, T, M, or V;F167 replaced with W, or Y; N168 replaced with Q; W169 replaced with F,or Y; Q170 replaced with N; H171 replaced with K, or R; N172 replacedwith Q; G173 replaced with A, I, L, S, T, M, or V; R174 replaced with H,or K; Q175 replaced with N; M176 replaced with A, G, I, L, S, T, or V;Y177 replaced with F, or W; V178 replaced with A, G, I, L, S, T, or M;A179 replaced with G, I, L, S, T, M, or V; L180 replaced with A, G, I,S, T, M, or V; N181 replaced with Q; G182 replaced with A, I, L, S, T,M, or V; K183 replaced with H, or R; G184 replaced with A, I, L, S, T,M, or V; A185 replaced with G, I, L, S, T, M, or V; R187 replaced withH, or K; R188 replaced with H, or K; G189 replaced with A, I, L, S, T,M, or V; Q190 replaced with N; K191 replaced with H, or R; T192 replacedwith A, G, I, L, S, M, or V; R193 replaced with H, or K; R194 replacedwith H, or K; K195 replaced with H, or R; N196 replaced with Q; T197replaced with A, G, I, L, S, M, or V; S198 replaced with A, G, I, L, T,M, or V; A199 replaced with G, I, L, S, T, M, or V; H200 replaced withK, or R; F201 replaced with W, or Y; L202 replaced with A, G, I, S, T,M, or V; M204 replaced with A, G, I, L, S, T, or V; V205 replaced withA, G, I, L, S, T, or M; V206 replaced with A, G, I, L, S, T, or M; H207replaced with K, or R; or S208 replaced with A, G, I, L, T, M, or V.

However, also preferred are KGF-2 molecules with nonconservative aminoacid substitutions, including: M1 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; W2 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; K3 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; W4 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; 15 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L6 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T7 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; H8 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; C9 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; A10 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; S1 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; A12 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;F13 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;P14 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; H15 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; L16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P17replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; G18 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C19 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; C20replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; C21 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or P; C22 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, or P; C23 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or P; F24 replaced with D, E, H, K, R, N, Q, A, G, I, L,S, T, M, V, P, or C; L25 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; L26 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L27replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F28 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L29 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V30 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S31 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; S32 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V33replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P34 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V35 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; T36 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; C37 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or P; Q38 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, F, W, Y, P, or C; A39 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L40 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G41 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q42replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;D43 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; M44 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V45 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S46 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P47 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; E48 replaced with H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; A49 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; T50 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; N51 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; S52 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S53replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S54 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; S55 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S56 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; F57 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; S58 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S59replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P60 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S61 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S62 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A63 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; G64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;R65 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;H66 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;V67 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R68 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S69 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; Y70 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; N71 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; H72 replaced with D, E,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L73 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; Q74 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, F, W, Y, P, or C; G75 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; D76 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; V77 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; R78 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; W79 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; R80 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; K81 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; L82 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F83replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S84replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F85 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T86 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; K87 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; Y88 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; F89 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; L90 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; K91 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; 192 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; E93 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; K94 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; N95 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; G96 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;K97 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;V98 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S99 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; G100 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T101 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; K102 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; K103 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; E104 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; N105 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, F, W, Y, P, or C; C106 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, or P; P107 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, or C; Y108 replaced with D, E, H, K, R, N, Q,A, G, I, L, S, T, M, V, P, or C; S109 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; I110 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; L111 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E112replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;I113 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T114 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S115 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; V16 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; E1 17 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; I118 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; G119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V120replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V121 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A122 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; V123 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; K124 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; A125 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I126replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N127 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S128 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; N129 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Y130 replaced with D, E,H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Y131 replaced with D, E,H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L132 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; A133 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; M134 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; N135 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; K136 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; K137 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; G138 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K139replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L140replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y141 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G142 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; S143 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; K144 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; E145 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; F146 replaced with D, E, H, K, R, N, Q, A, G,I, L, S, T, M, V, P, or C; N147 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, F, W, Y, P, or C; N148 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; D149 replaced with H, K, R, A, G,I, L,S, T, M, V, N, Q, F, W, Y, P, or C; C150 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, or P; K151 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L152 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; K153 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; E154 replaced with H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; R155 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; I156 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; E157 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; E158 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; N159 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; G160 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; Y161 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; N162 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,Y, P, or C; T163 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;Y164 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;A165 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S166 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; F167 replaced with D, E, H,K, R, N, Q, A, G11, L, S, T, M, V, P, or C; N168 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; W169 replaced with D, E,H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q170 replaced with D, E,H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; H171 replaced with D,E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N172 replaced with D,E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G173 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; R174 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; Q175 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; M176 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; Y177 replaced with D, E, H, K, R, N, Q, A, G,I, L, S, T, M, V, P, or C; V178 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; A179 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L180 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N181 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G182replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K183 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G184 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A185 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; P186 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; R187 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; R188 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; G189 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; Q190 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,F, W, Y, P, or C; K191 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; T192 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; R193 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; R194 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; K195 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; N196 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; T197 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S198replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A199 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; H200 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; F201 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; L202 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; P203 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; M204 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V205 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V206 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H207 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; or S208replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.

The substitution mutants can be tested in any of the assays describedherein for activity. Particularly preferred are KGF-2 molecules withconservative substitutions that maintain the activities and propertiesof the wild type protein; have an enhanced activity or property comparedto the wild type protein, while all other activities or properties aremaintained; or have more than one enhanced activity or property comparedto the wild type protein. In contrast, KGF-2 molecules withnonconservative substitutions preferably lack an activity or property ofthe wild type protein, while maintaining all other activities andproperties; or lack more than one activity or property of the wild typeprotein.

For example, activities or properties of KGF-2 that may be altered inKGF-2 molecules with conservative or nonconservative substitutionsinclude, but are not limited to: stimulation of growth of keratinocytes,epithelial cells, hair follicles, hepatocytes, renal cells, breasttissue, bladder cells, prostate cells, pancreatic cells; stimulation ofdifferentiation of muscle cells, nervous tissue, prostate cells, lungcells, hepatocytes, renal cells, breast tissue; promotion of woundhealing; angiogenesis stimulation; reduction of inflammation;cytoprotection; heparin binding; ligand binding; stability; solubility;and/or properties which affect purification.

Amino acids in KGF-2 that are essential for function can be identifiedby methods well known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, Science244:1081–1085 (1989)). The latter procedure introduces single alaninemutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity such as receptorbinding or in vitro and in vivo proliferative activity. (See, e.g.,Examples 10 and 11). Sites that are critical for ligand-receptor bindingcan also be determined by structural analysis such as crystallization,nuclear magnetic resonance or photoaffinity labelling. (See for example:Smith et al., J. Mol. Biol., 224: 899–904 (1992); and de Vos et al.Science, 255: 306–312 (1992).)

Another aspect of the present invention substitutions of serine forcysteine at amino acid positions 37 and 106 and 150. An uneven number ofcysteines means that at least one cysteine residue is available forintermolecular crosslinks or bonds that can cause the protein to adoptan undesirable tertiary structure. Novel KGF-2 proteins that have one ormore cysteine replaced by serine or e.g. alanine are generally purifiedat a higher yield of soluble, correctly folded protein. Although notproven, it is believed that the cysteine residue at position 106 isimportant for function. This cysteine residue is highly conserved amongall other FGF family members.

A further aspect of the present invention are fusions of KGF-2 withother proteins or fragments thereof such as fusions or hybrids withother FGF proteins, e.g. KGF (FGF-7), bFGF, aFGF, FGF-5, FGF-6, etc.Such a hybrid has been reported for KGF (FGF-7). In the published PCTapplication no.90/08771 a chimeric protein has been produced consistingof the first 40 amino acid residues of KGF and the C-terminal portion ofaFGF. The chimera has been reported to target keratinocytes like KGF,but lacked susceptibility to heparin, a characteristic of aFGF but notKGF. Fusions with parts of the constant domain of immunoglobulins (IgG)show often an increased half-life time in vivo. This has been shown,e.g., for chimeric proteins consisting of the first two domains of thehuman CD4-polypeptide with various domains of the constant regions ofthe heavy or light chains of mammalian immunoglobulins (European Patentapplication, Publication No.394 827, Traunecker et al., Nature 331:84–86(1988). Fusion proteins that have a disulfide-linked dimeric structurecan also be more efficient in binding monomeric molecules alone(Fountoulakis et al., J. of Biochemistry, 270: 3958–3964, (1995)).

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724–33 (1997); Harayama, Trends Biotechnol. 16(2):76–82(1998); Hansson, et al., J. Mol. Biol. 287:265–76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308–13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:1 and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antigenic/Hydrophilic Parts of KGF-2

As demonstrated in FIG. 4A–4E, there are 4 major highly hydrophilicregions in the KGF-2 protein. Amino acid residues Gly41-Asn 71,Lys91-Ser 109, Asn135-Tyr 164 and Asn 181-Ala 199 [SEQ ID NOS:25–28].There are two additional shorter predicted antigenic areas, Gln 74-Arg78 of FIG. 1 (SEQ ID NO:2) and Gln 170-Gln 175 of FIGS. 1A–1C (SEQ IDNO:2). Hydrophilic parts are known to be mainly at the outside (surface)of proteins and, therefore, available for antibodies recognizing theseregions. Those regions are also likely to be involved in the binding ofKGF-2 to its receptor(s). Synthetic peptides derived from these areascan interfere with the binding of KGF-2 to its receptor(s) and,therefore, block the function of the protein. Synthetic peptides fromhydrophilic parts of the protein may also be agonistic, i.e. mimic thefunction of KGF-2.

Thus, the present invention is further directed to isolated polypeptidescomprising a hydrophilic region of KGF-2 wherein said polypeptide is notmore than 150 amino acids in length, preferably not more than 100, 75,or 50 amino acids in length, which comprise one or more of the abovedescribed KGF-2 hydrophilic regions.

Epitope-Bearing Portions of KGF-2

In another aspect, the invention provides peptides and polypeptidescomprising epitope-bearing portions of the polypeptides of the presentinvention. These epitopes are immunogenic or antigenic epitopes of thepolypeptides of the present invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response in vivowhen the whole polypeptide of the present invention, or fragmentthereof, is the immunogen. On the other hand, a region of a polypeptideto which an antibody can bind is defined as an “antigenic determinant”or “antigenic epitope.” The number of in vivo immunogenic epitopes of aprotein generally is less than the number of antigenic epitopes. See,e.g., Geysen, et al., Proc. Natl. Acad. Sci. USA 81:3998–4002 (1983).However, antibodies can be made to any antigenic epitope, regardless ofwhether it is an immunogenic epitope, by using methods such as phagedisplay. See e.g., Petersen G. et al., Mol. Gen. Genet. 249:425–431(1995). Therefore, included in the present invention are bothimmunogenic epitopes and antigenic epitopes.

A list of exemplified amino acid sequences comprising immunogenicepitopes are shown in Table 1 below. It is pointed out that Table 1 onlylists amino acid residues comprising epitopes predicted to have thehighest degree of antigenicity using the algorithm of Jameson and Wolf,(1988) Comp. Appl. Biosci. 4:181–186 (said references incorporated byreference in their entireties). The Jameson-Wolf antigenic analysis wasperformed using the computer program PROTEAN, using default parameters(Version 3.11 for the Power MacIntosh, DNASTAR, Inc., 1228 South ParkStreet Madison, Wis.). Table 1 and portions of polypeptides not listedin Table 1 are not considered non-immunogenic. The immunogenic epitopesof Table 1 is an exemplified list, not an exhaustive list, because otherimmunogenic epitopes are merely not recognized as such by the particularalgorithm used. Amino acid residues comprising other immunogenicepitopes may be routinely determined using algorithms similar to theJameson-Wolf analysis or by in vivo testing for an antigenic responseusing methods known in the art. See, e.g., Geysen et al., supra; U.S.Pat. Nos. 4,708,781; 5,194,392; 4,433,092; and 5,480,971 (saidreferences incorporated by reference in their entireties).

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention. Non-limiting examples of antigenic polypeptides or peptidesthat can be used to KGF-2-specific antibodies include: a polypeptidecomprising amino acid residues in SEQ ID NO:2 from about Gly41-Asn71;Lys91-Ser109; Asn135-Tyr164; Asn181-Ala199; Gln74-Arg78; andGln170-Gln175. These polypeptide fragments have been determined to bearantigenic epitopes of the KGF-2 protein by the analysis of theJameson-Wolf antigenic index, as shown in FIG. 4, above.

It is particularly pointed out that the amino acid sequences of Table 1comprise immunogenic epitopes. Table 1 lists only the critical residuesof immunogenic epitopes determined by the Jameson-Wolf analysis. Thus,additional flanking residues on either the N-terminal, C-terminal, orboth — and C-terminal ends may be added to the sequences of Table 1 togenerate an epitope-bearing polypeptide of the present invention.Therefore, the immunogenic epitopes of Table 1 may include additionalN-terminal or C-terminal amino acid residues. The additional flankingamino acid residues may be contiguous flanking N-terminal and/orC-terminal sequences from the polypeptides of the present invention,heterologous polypeptide sequences, or may include both contiguousflanking sequences from the polypeptides of the present invention andheterologous polypeptide sequences. Polypeptides of the presentinvention comprising immunogenic or antigenic epitopes are at least 7amino acids residues in length. “At least” means that a polypeptide ofthe present invention comprising an immunogenic or antigenic epitope maybe 7 amino acid residues in length or any integer between 7 amino acidsand the number of amino acid residues of the full length polypeptides ofthe invention. Preferred polypeptides comprising immunogenic orantigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length.However, it is pointed out that each and every integer between 7 and thenumber of amino acid residues of the full length polypeptide areincluded in the present invention.

The immuno and antigenic epitope-bearing fragments may be specified byeither the number of contiguous amino acid residues, as described above,or further specified by N-terminal and C-terminal positions of thesefragments on the amino acid sequence of SEQ ID NO:2. Every combinationof a N-terminal and C-terminal position that a fragment of, for example,at least 7 or at least 15 contiguous amino acid residues in length couldoccupy on the amino acid sequence of SEQ ID NO:2 is included in theinvention. Again, “at least 7 contiguous amino acid residues in length”means 7 amino acid residues in length or any integer between 7 aminoacids and the number of amino acid residues of the full lengthpolypeptide of the present invention. Specifically, each and everyinteger between 7 and the number of amino acid residues of the fulllength polypeptide are included in the present invention.

Immunogenic and antigenic epitope-bearing polypeptides of the inventionare useful, for example, to make antibodies which specifically bind thepolypeptides of the invention, and in immunoassays to detect thepolypeptides of the present invention. The antibodies are useful, forexample, in affinity purification of the polypeptides of the presentinvention. The antibodies may also routinely be used in a variety ofqualitative or quantitative immunoassays, specifically for thepolypeptides of the present invention using methods known in the art.See, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press; 2nd Ed, Cold Spring Harbor, N.Y. (1988).

The epitope-bearing polypeptides of the present invention may beproduced by any conventional means for making polypeptides includingsynthetic and recombinant methods known in the art. For instance,epitope-bearing peptides may be synthesized using known methods ofchemical synthesis. For instance, Houghten has described a simple methodfor the synthesis of large numbers of peptides, such as 10–20 mgs of 248individual and distinct 13 residue peptides representing single aminoacid variants of a segment of the HA1 polypeptide, all of which wereprepared and characterized (by ELISA-type binding studies) in less thanfour weeks (Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131–5135(1985)). This “Simultaneous Multiple Peptide Synthesis (SMPS)” processis further described in U.S. Pat. No. 4,631,211 to Houghten andcoworkers (1986). In this procedure the individual resins for thesolid-phase synthesis of various peptides are contained in separatesolvent-permeable packets, enabling the optimal use of the manyidentical repetitive steps involved in solid-phase methods. A completelymanual procedure allows 500–1000 or more syntheses to be conductedsimultaneously (Houghten et al. (1985) Proc. Natl. Acad. Sci.82:5131–5135 at 5134).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347–2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carriers using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)resulting in chimeric polypeptides. Such fusion proteins may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84–86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958–3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or FLAG® tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972–897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Chemical Modifications

The KGF wild type and analogs may be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties may improve the solubility, the biological halflife or absorption of the protein. The moieties may also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 18th ed., Mack Publishing Co., Easton, Pa. (1990).Polyethylene glycol (PEG) is one such chemical moiety which has beenused for the preparation of therapeutic proteins. The attachment of PEGto proteins has been shown to protect against proteolysis, Sada et al.,J. Fermentation Bioengineering 71: 137–139 (1991). Various methods areavailable for the attachment of certain PEG moieties. For review, see:Abuchowski et al., in Enzymes as Drugs. (Holcerberg and Roberts, eds.)pp. 367–383 (1981). Many published patents describe derivatives of PEGand processes how to prepare them, e.g., Ono et al., U.S. Pat. No.5,342,940; Nitecki et al., U.S. Pat. No. 5,089,261; Delgado et al., U.S.Pat. No. 5,349,052. Generally, PEG molecules are connected to theprotein via a reactive group found on the protein. Amino groups, e.g. onlysines or the amino terminus of the protein are convenient for thisattachment among others.

The entire disclosure of each document cited in this section on“Polypeptides and Peptides” is hereby incorporated herein by reference.

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller et al., Nature, 310:105–111 (1984)). For example, apolypeptide corresponding to a fragment of a KGF-2 polypeptide can besynthesized by use of a peptide synthesizer. Furthermore, if desired,nonclassical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into the KGF-2 polypeptidesequence. Non-classical amino acids include, but are not limited to, tothe D-isomers of the common amino acids, 2,4-diaminobutyric acid,a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,b-alanine, fluoro-amino acids, designer amino acids such as b-methylamino acids, Ca-methyl amino acids, Na-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

The invention encompasses KGF-2 polypeptides which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited, to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028–1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:2, and/or an epitope, ofthe present invention (as determined by immunoassays well known in theart for assaying specific antibody-antigen binding). Antibodies of theinvention include, but are not limited to, polyclonal, monoclonal,multispecific, human, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies (including,e.g., anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. The term “antibody,” asused herein, refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site that immunospecifically binds anantigen. The immunoglobulin molecules of the invention can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60–69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547–1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Preferred epitopes of theinvention include: amino acids 41–71, 91–109, 135–164, 181–199, 74–78,and 170–175 of SEQ ID NO:2, as well as polynucleotides that encode theseepitopes. Antibodies which specifically bind any epitope or polypeptideof the present invention may also be excluded. Therefore, the presentinvention includes antibodies that specifically bind polypeptides of thepresent invention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologs of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻²M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴M, 10⁴M, 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁷M, 5×10⁻⁸ M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹ M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10¹¹M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴M, 10⁻¹⁴ M, 5×10⁻¹⁵M, or 10⁻¹⁵M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated byreference in the entirety).

The antibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. The term “monoclonal antibody” is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced. Monoclonal antibodies can be prepared using a wide varietyof techniques known in the art including the use of hybridoma,recombinant, and phage display technology.

Hybridoma techniques include those known in the art and taught in Harlowet al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: MONOCLONAL ANTIBODIES ANDT-CELL HYBRIDOMAS 563–681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). Fab and F(ab′)2fragments may be produced by proteolytic cleavage, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments).

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA and phage display technologyor through synthetic chemistry using methods known in the art. Forexample, the antibodies of the present invention can be prepared usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface of aphage particle which carries polynucleotide sequences encoding them.Phage with a desired binding property are selected from a repertoire orcombinatorial antibody library (e.g. human or murine) by selectingdirectly with antigen, typically antigen bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage including fd and M13 with Fab, Fv or disulfide stabilized Fvantibody domains recombinantly fused to either the phage gene III orgene VIII protein. Examples of phage display methods that can be used tomake the antibodies of the present invention include those disclosed inBrinkman U. et al. (1995) J. Immunol. Methods 182:41–50; Ames, R. S. etal. (1995) J. Immunol. Methods 184:177–186; Kettleborough, C. A. et al.(1994) Eur. J. Immunol. 24:952–958; Persic, L. et al. (1997) Gene187:9–18; Burton, D. R. et al. (1994) Advances in Immunology 57:191–280;PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236;WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637,5,780,225, 5,658,727 and 5,733,743 (saidreferences incorporated by reference in their entireties).

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in WO 92/22324; Mullinax, R. L. et al. (1992)BioTechniques 12(6):864–869; and Sawai, H. et al. (1995) AJRI 34:26–34;and Better, M. et al. (1988) Science 240:1041–1043 (said referencesincorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991) Methods in Enzymology 203:46–88; Shu, L.et al. (1993) PNAS 90:7995–7999; and Skerra, A. et al. (1988) Science240:1038–1040. For some uses, including in vivo use of antibodies inhumans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies, S. D. et al.(1989) J. Immunol. Methods 125:191–202; and U.S. Pat. No. 5,807,715.Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. No. 5,530,101; and5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; PadlanE. A., (1991) Molecular Immunology 28(4/5):489–498; Studnicka G. M. etal. (1994) Protein Engineering 7(6):805–814; Roguska M. A. et al. (1994)PNAS 91:969–973), and chain shuffling (U.S. Pat. No. 5,565,332). Humanantibodies can be made by a variety of methods known in the artincluding phage display methods described above. See also, U.S. Pat.Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO91/10741 (said references incorporated by reference in theirentireties).

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferrably, antibodies of the present invention bind anantigenic epitope disclosed herein, or a portion thereof. The inventionfeatures both receptor-specific antibodies and ligand-specificantibodies. The invention also features receptor-specific antibodieswhich do not prevent ligand binding but prevent receptor activation.Receptor activation (i.e., signaling) may be determined by techniquesdescribed herein or otherwise known in the art. For example, receptoractivation can be determined by detecting the phosphorylation (e.g.,tyrosine or serine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981–1988 (1998); Chenet al., Cancer Res. 58(16):3668–3678 (1998); Harrop et al., J. Immunol.161(4):1786–1794 (1998); Zhu et al., Cancer Res. 58(15):3209–3214(1998); Yoon et al., J. Immunol. 160(7):3170–3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237–247 (1998); Pitard et al., J. Immunol.Methods 205(2):177–190 (1997); Liautard et al., Cytokine 9(4):233–241(1997); Carlson et al., J. Biol. Chem. 272(17):11295–11301 (1997);Taryman et al., Neuron 14(4):755–762 (1995); Muller et al., Structure6(9):1153–1167 (1998); Bartunek et al., Cytokine 8(1):14–20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety). In a preferred embodiment, levels of KGF-2 aredetected in a purified sample using goat and chicken antibodies (seeexample 50, below).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563–681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples. In a non-limiting example, mice canbe immunized with a polypeptide of the invention or a cell expressingsuch peptide. Once an immune response is detected, e.g., antibodiesspecific for the antigen are detected in the mouse serum, the mousespleen is harvested and splenocytes isolated. The splenocytes are thenfused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC®. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41–50(1995); Ames et al., J. Immunol. Methods 184:177–186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952–958 (1994); Persic et al.,Gene 187:9–18 (1997); Burton et al., Advances in Immunology 57:191–280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864–869(1992); and Sawai et al., AJRI 34:26–34 (1995); and Better et al.,Science 240:1041–1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46–88 (1991); Shu etal., PNAS 90:7995–7999 (1993); and Skerra et al., Science 240:1038–1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191–202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species antibody that binds the desired antigenhaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework regions from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489–498(1991); Studnicka et al., Protein Engineering 7(6):805–814 (1994);Roguska. et al., PNAS 91:969–973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65–93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No.0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and GenPharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899–903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437–444;(1989) and Nissinoff, J. Immunol. 147(8):2429–2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

The invention further relates to antibodies that act as agonists orantagonists of the polypeptides of the present invention. Antibodieswhich act as agonists or antagonists of the polypeptides of the presentinvention include, for example, antibodies which disrupt receptor/ligandinteractions with the polypeptides of the invention either partially orfully. For example, the present invention includes antibodies thatdisrupt the ability of the proteins of the invention to multimerize. Inanother example, the present invention includes antibodies which allowthe proteins of the invention to multimerize, but disrupts the abilityof the proteins of the invention to bind one or more KGF-2receptor(s)/ligand(s). In yet another example, the present inventionincludes antibodies which allow the proteins of the invention tomultimerize, and bind KGF-2 receptor(s)/ligand(s), but blocks biologicalactivity associated with the KGF-2/receptor/ligand complex.

Antibodies which act as agonists or antagonists of the polypeptides ofthe present invention also include, both receptor-specific antibodiesand ligand-specific antibodies. Included are receptor-specificantibodies that do not prevent ligand binding but prevent receptoractivation. Receptor activation (i.e., signaling) may be determined bytechniques described herein or otherwise known in the art. Also includedare receptor-specific antibodies which both prevent ligand binding andreceptor activation. Likewise, included are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included are antibodies that activate the receptor. Theseantibodies may act as agonists for either all or less than all of thebiological activities affected by ligand-mediated receptor activation.The antibodies may be specified as agonists or antagonists forbiological activities comprising specific activities disclosed herein.The above antibody agonists can be made using methods known in the art.See e.g., WO 96/40281; U.S. Pat. No. 5,811,097; Deng, B. et al., Blood92(6):1981–1988 (1998); Chen, Z. et al., Cancer Res. 58(16):3668–3678(1998); Harrop, J. A. et al., J. Immunol. 161(4): 1786–1794 (1998); Zhu,Z. et al., Cancer Res. 58(15):3209–3214 (1998); Yoon, D. Y. et al., J.Immunol. 160(7):3170–3179 (1998); Prat, M. et al., J. Cell. Sci.111(Pt2):237–247 (1998); Pitard, V. et al., J. Immunol. Methods205(2):177–190 (1997); Liautard, J. et al., Cytokinde 9(4):233–241(1997); Carlson, N. G. et al., J. Biol. Chem. 272(17):11295–11301(1997); Taryman, R. E. et al., Neuron 14(4):755–762 (1995); Muller, Y.A. et al., Structure 6(9):1153–1167 (1998); Bartunek, P. et al.,Cytokine 8(1):14–20 (1996) (said references incorporated by reference intheir entireties).

As discussed above, antibodies to the KGF-2 proteins of the inventioncan, in turn, be utilized to generate anti-idiotype antibodies that“mimic” KGF-2 using techniques well known to those skilled in the art.(See, e.g., Greenspan & Bona, FASEB J. 7(5):437–444; (1989) andNissinoff, J. Immunol. 147(8):2429–2438 (1991)). For example, antibodieswhich bind to KGF-2 and competitively inhibit KGF-2 multimerizationand/or binding to ligand can be used to generate anti-idiotypes that“mimic” the KGF-2 multimerization and/or binding domain and, as aconsequence, bind to and neutralize KGF-2 and/or its ligand. Suchneutralizing anti-idiotypes or Fab fragments of such anti-idiotypes canbe used in therapeutic regimens to neutralize KGF-2 ligand. For example,such anti-idiotypic antibodies can be used to bind KGF-2, or to bindKGF-2 ligands/receptors, and thereby block KGF-2 biological activity.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278:457–479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851–855 (1984);Neuberger et al., Nature 312:604–608 (1984); Takeda et al., Nature314:452–454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423–42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879–5883 (1988); and Wardet al., Nature 334:544–54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038–1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101–3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503–5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355–359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51–544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERA, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1–2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418(Goldspiel et al., Clinical Pharmacy 12:488–505 (1993); Wu and Wu,Biotherapy 3:87–95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol.32:573–596 (1993); Mulligan, Science 260:926–932 (1993); and Morgan andAnderson, Ann. Rev. Biochem. 62:191–217 (1993); May, 1993, TIB TECH11(5):155–215); and hygro, which confers resistance to hygromycin(Santerre et al., Gene 30:147 (1984)). Methods commonly known in the artof recombinant DNA technology may be routinely applied to select thedesired recombinant clone, and such methods are described, for example,in Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley& Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981),which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91–99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428–1432 (1992); Fell et al., J. Immunol.146:2446–2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535–10539 (1991); Zheng et al., J. Immunol.154:5590–5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337–11341(1992) (said references incorporated by reference in theirentireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:2 may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. Further, the polypeptides corresponding to SEQ ID NO:2 maybe fused or conjugated to the above antibody portions to facilitatepurification. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature331:84–86 (1988). The polypeptides of the present invention fused orconjugated to an antibody having disulfide-linked dimeric structures(due to the IgG) may also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958–3964 (1995)). In manycases, the Fc part in a fusion protein is beneficial in therapy anddiagnosis, and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5 receptor, havebeen fused with Fc portions for the purpose of high-throughput screeningassays to identify antagonists of hIL-5. (See, Bennett et al., J.Molecular Recognition 8:52–58 (1995); Johanson et al., J. Biol. Chem.270:9459–9471 (1995).)

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821–824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “FLAG®” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, ²¹³Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-α, TNF-β, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567–1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243–56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.623–53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475–506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp.303–16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119–58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737–49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays For Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1–4 hours) at 4° C, adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%–20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1. The binding affinity ofan antibody to an antigen and the off-rate of an antibody-antigeninteraction can be determined by competitive binding assays. One exampleof a competitive binding assay is a radioimmunoassay comprising theincubation of labeled antigen (e.g., ³H or ¹²⁵I with the antibody ofinterest in the presence of increasing amounts of unlabeled antigen, andthe detection of the antibody bound to the labeled antigen. The affinityof the antibody of interest for a particular antigen and the bindingoff-rates can be determined from the data by scatchard plot analysis.Competition with a second antibody can also be determined usingradioimmunoassays. In this case, the antigen is incubated with antibodyof interest conjugated to a labeled compound (e.g., ³H or ¹²⁵I) in thepresence of increasing amounts of an unlabeled second antibody.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of KGF-2polypeptides or fragments thereof by recombinant techniques.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention. Thepresent invention also relates to vectors which include polynucleotidesof the present invention, host cells which are genetically engineeredwith vectors of the invention and the production of polypeptides of theinvention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the KGF-2 genes. The culture conditions,such as temperature, pH and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequences) (promoter) to direct cDNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli, lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC®Accession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; adenoviruses and plant cells. Appropriate culturemediums and conditions for the above-described host cells are known inthe art.

In addition to the use of expression vectors in the practice of thepresent invention, the present invention further includes novelexpression vectors comprising operator and promoter elements operativelylinked to nucleotide sequences encoding a protein of interest. Oneexample of such a vector is pHE4-5 which is described in detail below.

As summarized in FIGS. 50 and 51, components of the pHE4-5 vector (SEQID NO:147) include: 1) a neomycinphosphotransferase gene as a selectionmarker, 2) an E. coli origin of replication, 3) a T5 phage promotersequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence,6) the lactose operon repressor gene (lacIq). The origin of replication(oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promotersequence and operator sequences were made synthetically. Syntheticproduction of nucleic acid sequences is well known in the art. CLONTECH95/96 Catalog, pages 215–216, CLONTECH, 1020 East Meadow Circle, PaloAlto, Calif. 94303. A nucleotide sequence encoding KGF-2 (SEQ ID NO:1),is operatively linked to the promoter and operator by inserting thenucleotide sequence between the NdeI and Asp718 sites of the pHE4-5vector.

As noted above, the pHE4-5 vector contains a lacIq gene. LacIq is anallele of the lacI gene which confers tight regulation of the lacoperator. Amann, E. et al., Gene 69:301–315 (1988); Stark, M., Gene51:255–267 (1987). The lacIq gene encodes a repressor protein whichbinds to lac operator sequences and blocks transcription of down-stream(i.e., 3′) sequences. However, the lacIq gene product dissociates fromthe lac operator in the presence of either lactose or certain lactoseanalogs, e.g., isopropyl B-D-thiogalactopyranoside (IPTG). KGF-2 thus isnot produced in appreciable quantities in uninduced host cellscontaining the pHE4-5 vector. Induction of these host cells by theaddition of an agent such as IPTG, however, results in the expression ofthe KGF-2 coding sequence.

The promoter/operator sequences of the pHE4-5 vector (SEQ ID NO:148)comprise a T5 phage promoter and two lac operator sequences. Oneoperator is located 5′ to the transcriptional start site and the otheris located 3′ to the same site. These operators, when present incombination with the lacIq gene product, confer tight repression ofdown-stream sequences in the absence of a lac operon inducer, e.g.,IPTG. Expression of operatively linked sequences located down-streamfrom the lac operators may be induced by the addition of a lac operoninducer, such as IPTG. Binding of a lac inducer to the lacIq proteinsresults in their release from the lac operator sequences and theinitiation of transcription of operatively linked sequences. Lac operonregulation of gene expression is reviewed in Devlin, T., TEXTBOOK OFBIOCHEMISTRY WITH CLINICAL CORRELATIONS, 4th Edition (1997), pages802–807.

The pHE4 series of vectors contain all of the components of the pHE4-5vector except for the KGF-2 coding sequence. Features of the pHE4vectors include optimized synthetic T5 phage promoter, lac operator, andShine-Delagarno sequences. Further, these sequences are also optimallyspaced so that expression of an inserted gene may be tightly regulatedand high level of expression occurs upon induction.

Among known bacterial promoters suitable for use in the production ofproteins of the present invention include the E. coli lacI and lacZpromoters, the T3 and T7 promoters, the gpt promoter, the lambda PR andPL promoters and the trp promoter. Suitable eukaryotic promoters includethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous Sarcoma Virus (RSV), and metallothionein promoters,such as the mouse metallothionein-I promoter.

The pHE4-5 vector also contains a Shine-Delgarno sequence 5′ to the AUGinitiation codon. Shine-Delgarno sequences are short sequences generallylocated about 10 nucleotides up-stream (i.e., 5′) from the AUGinitiation codon. These sequences essentially direct prokaryoticribosomes to the AUG initiation codon.

Thus, the present invention is also directed to expression vector usefulfor the production of the proteins of the present invention. This aspectof the invention is exemplified by the pHE4-5 vector (SEQ ID NO:147).The pHE4-5 vector containing a cDNA insert encoding KGF-2 Δ33 wasdeposited at the ATCC® on Jan. 9, 1998 as ATCC® No. 209575.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPA0815 (all available from Invitrogen, Carlbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that KGF-2 polypeptidesmay in fact be expressed by a host cell lacking a recombinant vector.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L. et al., Basic Methods inMolecular Biology (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizereceptors. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,shIL5-receptor has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., J. Mol. Recognition, Vol. 8 52–58 (1995) and K.Johanson et al., J. Biol. Chem., 270(16):9459–9471 (1995).

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC® 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C 127, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

KGF-2 polypeptides can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

KGF-2 polypeptides, and preferably the secreted form, can also berecovered from: products purified from natural sources, including bodilyfluids, tissues and cells, whether directly isolated or cultured;products of chemical synthetic procedures; and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect, and mammaliancells. Depending upon the host employed in a recombinant productionprocedure, the KGF-2 polypeptides may be glycosylated or may benon-glycosylated. In addition, KGF-2 polypeptides may also include aninitial modified methionine residue, in some cases as a result ofhost-mediated processes. Thus, it is well known in the art that theN-terminal methionine encoded by the translation initiation codongenerally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins, this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

In one embodiment, the yeast Pichia pastoris is used to express KGF-2protein in a eukaryotic system. Pichia pastoris is a methylotrophicyeast which can metabolize methanol as its sole carbon source. A mainstep in the methanol metabolization pathway is the oxidation of methanolto formaldehyde using O₂. This reaction is catalyzed by the enzymealcohol oxidase. In order to metabolize methanol as its sole carbonsource, Pichia pastoris must generate high levels of alcohol oxidasedue, in part, to the relatively low affinity of alcohol oxidase for O₂.Consequently, in a growth medium depending on methanol as a main carbonsource, the promoter region of one of the two alcohol oxidase genes(AOX1) is highly active. In the presence of methanol, alcohol oxidaseproduced from the AOX1 gene comprises up to approximately 30% of thetotal soluble protein in Pichia pastoris. See, Ellis, S. B., et al.,Mol. Cell. Biol. 5:1111–21 (1985); Koutz, P. J, et al., Yeast 5:167–77(1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859–76 (1987).Thus, a heterologous coding sequence, such as, for example, a KGF-2polynucleotide of the present invention, under the transcriptionalregulation of all or part of the AOX1 regulatory sequence is expressedat exceptionally high levels in Pichia yeast grown in the presence ofmethanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding a KGF-2 polypeptide of the invention, as set forth herein, in aPichia yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a KGF-2 protein of the invention by virtueof the strong AOX1 promoter linked to the Pichia pastoris alkalinephosphatase (PHO) secretory signal peptide (i.e., leader) locatedupstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHI-D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

In another embodiment, high-level expression of a heterologous codingsequence, such as, for example, a KGF-2 polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., KGF-2 coding sequence), and/or to includegenetic material (e.g., heterologous polynucleotide sequences) that isoperably associated with KGF-2 polynucleotides of the invention, andwhich activates, alters, and/or amplifies endogenous KGF-2polynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous KGF-2 polynucleotide sequences via homologousrecombination, resulting in the formation of a new transcription unit(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No.5,733,761, issued Mar. 31, 1998; International Publication No. WO96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932–8935 (1989); and Zijlstra et al., Nature 342:435–438 (1989),the disclosures of each of which are incorporated by reference in theirentireties).

Diagnostic and Therapeutic Applications of KGF-2

As used in the section below, “KGF-2” is intended to refer to thefull-length and mature forms of KGF-2 described herein and to the KGF-2analogs, derivatives and mutants described herein. This invention isalso related to the use of the KGF-2 gene as part of a diagnostic assayfor detecting diseases or susceptibility to diseases related to thepresence of mutations in the KGF-2 nucleic acid sequences.

Individuals carrying mutations in the KGF-2 gene may be detected at theDNA level by a variety of techniques. Nucleic acids for diagnosis may beobtained from a patient's cells, such as from blood, urine, saliva,tissue biopsy and autopsy material. The genomic DNA may be used directlyfor detection or may be amplified enzymatically by using PCR (Saiki etal., Nature 324:163–166 (1986)) prior to analysis. RNA or cDNA may alsobe used for the same purpose. As an example, PCR primers complementaryto the nucleic acid encoding KGF-2 can be used to identify and analyzeKGF-2 mutations. For example, deletions and insertions can be detectedby a change in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled KGF-2 RNA or alternatively, radiolabeled KGF-2 antisenseDNA sequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397–4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of KGF-2 protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease, for example, a tumor. Assays used to detect levels of KGF-2protein in a sample derived from a host are well-known to those of skillin the art and include radioimmunoassays, competitive-binding assays,Western Blot analysis, ELISA assays and “sandwich” assays. An ELISAassay (Coligan, et al., Current Protocols in Immunology, 1(2), Chapter6, (1991)) initially comprises preparing an antibody specific to theKGF-2 antigen, preferably a monoclonal antibody. In addition a reporterantibody is prepared against the monoclonal antibody. To the reporterantibody is attached a detectable reagent such as radioactivity,fluorescence or, in this example, a horseradish peroxidase enzyme. Asample is removed from a host and incubated on a solid support, e.g. apolystyrene dish, that binds the proteins in the sample. Any freeprotein binding sites on the dish are then covered by incubating with anon-specific protein like bovine serum albumen. Next, the monoclonalantibodies attach to any KGF-2 proteins attached to the polystyrenedish. All unbound monoclonal antibody is washed out with buffer. Thereporter antibody linked to horseradish peroxidase is now placed in thedish resulting in binding of the reporter antibody to any monoclonalantibody bound to KGF-2. Unattached reporter antibody is then washedout. Peroxidase substrates are then added to the dish and the amount ofcolor developed in a given time period is a measurement of the amount ofKGF-2 protein present in a given volume of patient sample when comparedagainst a standard curve.

A competition assay may be employed wherein antibodies specific to KGF-2are attached to a solid support and labeled KGF-2 and a sample derivedfrom the host are passed over the solid support and the amount of labeldetected, for example by liquid scintillation chromatography, can becorrelated to a quantity of KGF-2 in the sample.

A “sandwich” assay is similar to an ELISA assay. In a “sandwich” assayKGF-2 is passed over a solid support and binds to antibody attached to asolid support. A second antibody is then bound to the KGF-2. A thirdantibody which is labeled and specific to the second antibody is thenpassed over the solid support and binds to the second antibody and anamount can then be quantified.

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler & Milstein, Nature,256:495–497 (1975)), the trioma technique, the human B-cell hybridomatechnique (Kozbor, et al., Immunology Today 4:72 (1983)), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77–96 (1985)).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The polypeptides of the present invention have been shown to stimulategrowth of epithelium. Thus, the polypeptides of the present inventionmay be employed to stimulate growth of epithelium. “Epithelium” refersto the covering of internal and external surfaces of the body, includingthe lining of vessels and other small cavities. It consists of cellsjoined by small amounts of cementing substances. Epithelium isclassified into types on the basis of the number of layers deep and theshape of the superficial cells. Epithelial cells include anteriuscorneae, Barrett's epithelium, capsular epithelium, ciliated epithelium,columnar epithelium, corneal epithelium, cubical epithelium, epitheliumductus semicircularis, enamel epithelium, false epithelium, germinalepithelium, gingival epithelium, glandular epithelium, glomerularepithelium, laminated epithelium, epithelium of the lend, mesenchymalepithelium, olfactory epithelium, pavement epithelium, pigmentaryepithelium, protective epithelium, pseudostratified epithelium,pyramidal epithelium, respiratory epithelium, rod epithelium,seminiferous epithelium, sensory epithelium, simple epithelium, squamousepithelium, stratified epithelium, subcapsular epithelium, sulcularepithelium, tessellated epithelium, transitional epithelium, andepithelial cells of the eye, tongue, glands, oral mucosa, duodenum,ileum, jejunum, cecum, nasal passages, esophagus, colon, mammary glands,and the female and male reproductive systems.

“Glands” refer to an aggregation of cells, specialized to secrete orexcrete materials not related to their ordinary metabolic needs.Examples of glands which may include epithelial cells include: absorbentclangs, accessory glands, acinar glands, acid glands, admaxillaryglands, adrenal glands, aggregate glands, Albarran's gland, anal glands,alveolar glands, anteprostatic glands, aortic glands, apical glands ofthe tongue, apocrine glands, areolar glands, arterial glands,arteriococcygeal glands, arytenoid glands, Aselli's glands, Avicenna'sglands, atribiliary gland, axillary glands, Bartholin's glands, Bauhin'sglands, Baumgarten's glands, glands of the biliary mucosa, Blandin'sglands, blood vessel glands, Boerhaave's glands, Bonnot's glands,Bowman's glands, brachial glands, bronchial glands, Bruch's glands,Brunner's glands, buccal glands, bulbocavernous glands, cardiac glands,carotid glands, celiac glands, ceruminous glands, cervical glands of theuterus, choroid glands, Ciaccio's glands, ciliary glands of theconjunctiva, circumanal glands, Cloquet's glands, Cobelli's glands,coccygeal glands, coil glands, compound glands, conglobate gland,conjunctival glands, Cowper's gland, cutaneous glands, cytogenic glands,ductless glands, duodenal glands, Duverney's gland, Ebner's gland,eccrine glands, Eglis' glands, endocrine glands, endoepithelial glands,esophageal glands, excretory glands, exocrine glands, follicular glandsof the duct, fundus glands, gastric glands, gastroepiploic glands,glands of Gay, genital glands, gingival glands, Gley's glands, globateglands, glomerate glands, glossopalatine glands, Guerin's glands,guttural glands, glands of Haller, Harder's glands, haversian glands,hedonic glands, hemal glands, hemal lymph glands, hematopoietic glands,hemolymph glands, Henle's glands, hepatic glands, heterocrine glands,hibernating glands, holocrine glands and incretory glands.

Further examples of glands include intercarotid glands, intermediateglands, interscapular glands, interstitial glands, intestinal glands,intraepithelial glands, intramuscular glands of the tongue, jugulargland, Krause's glands, labial glands of the mouth, lacrimal glands,accessory lacrimal glands, lactiferous gland, glands of the largeintestine, large sweat glands, laryngeal glands, lenticular glands ofthe stomach and tongue, glands of Lieberkuhn, lingual glands, anteriorlingual glands, Littre's glands, Luschka's gland, lymph glands,extraparotid lymph glands, malar glands, mammary glands, accessorymammary glands, mandibular glands, Manz' glands, Mehlis' glands,meibomian glands, merocrine glands, mesenteric glands, mesocolic glands,mixed glands, molar glands, Moll's glands, monoptyphic glands,Montgomery's glands, Morgagni's glands, glands of the mouth,mucilaginous glands, muciparous glands, mucous glands, lingual mucousglands, mucous glands of the auditory tube, mucous glands of theduodenum, mucous glands of the eustachian tube, multicellular glands,myometrial glands, Naboth's glands, nabothian glands, nasal glands,glands of the neck, odoriferous glands of the prepuce, oil glands,olfactory glands, oxyntic glands, pacchionian glands, palatine glands,pancreaticosplenic glands, parafrenal glands, parathyroid glands,parurethral glands, parotid glands, accessory parotid glands, pectoralglands, peptic glands, perspiratory glands, Peyre's glands, pharyngealglands, Philip's glands, pineal glands, and pituitary.

Other examples of glands include Poirier's glands, polyptychich glands,preen gland, pregnancy glands, prehyoid glands, preputial glands,prostate gland, puberty glands, pyloric glands, racemose glands,retrolingual glands, retromolar glands, Rivinus gland, Rosenmullergland, saccular gland, salivary glands, abdominal salivary glands,external salivary glands, internal salivary glands, Sandstrom's glands,Schuller's glands, sebaceous glands, sebaceous glands of theconjunctiva, sentinal glands, seromucous glands, serous glands, Serres'glands, Sigmunds glands, Skene's glands, simple gland, glands of thesmall intestine, solitary glands of the large intestine, splenoid gland,Stahr's gland, staplyline glands, subauricular glands, sublingualglands, submandibular glands, suboriferous glands, suprarenal glands,accessory suprarenal glands, Suzanne's gland, sweat glands, synovialglands, tarsal glands, Theile's glands, thymus gland, thyroid gland,accessory thyroid glands, glands of the tongue, tracheal glands, tachomaglands, tubular glands, tubuloacinar glands, tympanic glands, glands ofTyson, unicellular glands, urethral glands, urethral glands of thefemale urethra, uropygial gland, uterine glands, utricular glands,vaginal glands, vascular glands, vestibular glands (greater and lesser),Virchow's gland, vitelline gland, bulbovaginal gland, Waldeyer's glands,Weber's glands, glands of Wolfring, glands of Zeis and Zuckerkandl'sglands.

Thus, KGF-2 may be employed to stimulate the growth of any of thesecells or cells within these glands.

The polypeptides of the present invention may be employed to stimulatenew blood vessel growth or angiogenesis. Particularly, the polypeptidesof the present invention may stimulate keratinocyte cell growth andproliferation. Accordingly the present invention provides a process forutilizing such polypeptides, or polynucleotides encoding suchpolypeptides for therapeutic purposes, for example, to stimulateepithelial cell proliferation and basal keratinocytes for the purpose ofwound healing, and to stimulate hair follicle production and healing ofdermal wounds.

As noted above, the polypeptides of the present invention may beemployed to heal dermal wounds by stimulating epithelial cellproliferation. These wounds may be of superficial nature or may be deepand involve damage of the dermis and the epidermis of skin. Thus, thepresent invention provides a method for the promotion of wound healingthat involves the administration of an effective amount of KGF-2 to anindividual.

The individual to which KGF-2 is administered may heal wounds at anormal rate or may be healing impaired. When administered to anindividual who is not healing impaired, KGF-2 is administered toaccelerate the normal healing process. When administered to anindividual who is healing impaired, KGF-2 is administered to facilitatethe healing of wounds which would otherwise heal slowly or not at all.As noted below, a number of afflictions and conditions can result inhealing impairment. These afflictions and conditions include diabetes(e.g., Type II diabetes mellitus), treatment with both steroids andother pharmacological agents, and ischemic blockage or injury. Steroidswhich have been shown to impair wound healing include cortisone,hydrocortisone, dexamethasone, and methylprednisolone.

Non-steroid compounds, e.g., octreotide acetate, have also been shown toimpair wound healing. Waddell, B. et al., Am. Surg. 63:446–449 (1997).The present invention is believed to promote wound healing inindividuals undergoing treatment with such non-steroid agents.

A number of growth factors have been shown to promote wound healing inhealing impaired individuals. See, e.g., Steed, D. et al., J. Am. Coll.Surg. 183:61–64 (1996); Richard, J. et al., Diabetes Care 18: 64–69(1995); Steed, D., J. Vasc. Surg. 21:71–78 (1995); Kelley, S. et al.,Proc. Soc. Exp. Biol. 194:320–326 (1990). These growth factors includegrowth hormone-releasing factor, platelet-derived growth factor, andbasic fibroblast growth factor. Thus, the present invention alsoencompasses the administration of KGF-2 in conjunction with one or moreadditional growth factors or other agent which promotes wound healing.

The present invention also provides a method for promoting the healingof anastomotic and other wounds caused by surgical procedures inindividuals which both heal wounds at a normal rate and are healingimpaired. This method involves the administration of an effective amountof KGF-2 to an individual before, after, and/or during anastomotic orother surgery. Anastomosis is the connecting of two tubular structures,as which happens, for example, when a mid-section of intestine isremoved and the remaining portions are linked together to reconstitutethe intestinal tract. Unlike with cutaneous healing, the healing processof anastomotic wounds is generally obscured from view. Further, woundhealing, at least in the gastrointestinal tract, occurs rapidly in theabsence of complications; however, complications often requirecorrection by additional surgery. Thornton, F. and Barbul, A., Surg.Clin. North Am. 77:549–573 (1997). As shown in Examples 21 and 28,treatment with KGF-2 causes a significant decrease in peritoneal leakageand anastomotic constriction following colonic anastomosis. KGF-2 isbelieved to cause these results by accelerating the healing process thusdecreasing the probability of complications arising following suchprocedures.

Thus, the present invention also provides a method for acceleratinghealing after anastomoses or other surgical procedures in an individual,which heals wounds at a normal rate or is healing impaired, compromisingthe administration of an effective amount of KGF-2.

The polypeptides of the present invention may also be employed tostimulate differentiation of cells, for example muscle cells, cellswhich make up nervous tissue, prostate cells, and lung cells.

KGF-2 may be clinically useful in stimulating healing of woundsincluding surgical wounds, excisional wounds, deep wounds involvingdamage of the dermis and epidermis, eye tissue wounds, dental tissuewounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitusulcers, arterial ulcers, venous stasis ulcers, and burns resulting fromheat exposure or chemicals, in normal individuals and those subject toconditions which induce abnormal wound healing such as uremia,malnutrition, vitamin deficiencies, obesity, infection,immunosuppression and complications associated with systemic treatmentwith steroids, radiation therapy, and antineoplastic drugs andantimetabolites. KGF-2 is also useful for promoting the healing ofwounds associated with ischemia and ischemic injury, e.g., chronicvenous leg ulcers caused by an impairment of venous circulatory systemreturn and/or insufficiency.

KGF-2 can also be used to promote dermal reestablishment subsequent todermal loss. In addition, KGF-2 can be used to increase the tensilestrength of epidermis and epidermal thickness.

KGF-2 can be used to increase the adherence of skin grafts to a woundbed and to stimulate re-epithelialization from the wound bed. Thefollowing are types of grafts that KGF-2 could be used to increaseadherence to a wound bed: autografts, artificial skin, allografts,autodermic graft, autoepidermic grafts, avacular grafts, Blair-Browngrafts, bone graft, brephoplastic grafts, cutis graft, delayed graft,dermic graft, epidermic graft, fascia graft, full thickness graft,heterologous graft, xenograft, homologous graft, hyperplastic graft,lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft,omenpal graft, patch graft, pedicle graft, penetrating graft, split skingraft, thick split graft. KGF-2 can be used to promote skin strength andto improve the appearance of aged skin.

It is believed that KGF-2 will also produce changes in hepatocyteproliferation, and epithelial cell proliferation in the lung, breast,pancreas, stomach, small intestine, and large intestine. KGF-2 canpromote proliferation of epithelial cells such as sebocytes, hairfollicles, hepatocytes, type II pneumocytes, mucin-producing gobletcells, and other epithelial cells and their progenitors contained withinthe skin, lung, liver, kidney and gastrointestinal tract. As shown inExample 31, KGF-2 stimulates the proliferation of hepatocytes. Thus,KGF-2 can also be used prophylactically or therapeutically to prevent orattenuate acute or chronic viral hepatitis as well as fulminant orsubfulminant liver failure caused by diseases such as acute viralhepatitis, cirrhosis, drug- and toxin-induced hepatitis (e.g,acetaminophen, carbon tetrachloride, methotrexate, organic arsenicals,and other hepatotoxins known in the art), autoimmune chronic activehepatitis, liver transplantation, and partial hepatectomy (Cotran et al.Pathologic basis of disease. (5^(th) ed). Philadelphia, W. B. SaundersCompany,1994). KGF-2 can also be used to stimulate or promote liverregeneration and in patients with alcoholic liver disease. KGF-2 can beused to treat fibrosis of the liver.

Approximately 80% of acute pancreatitis cases are associated withbiliary tract disease and alcoholism (Rattner D. W., Scand JGastroenterol 31:6–9 (1996); Cotran et al. Pathologic basis of disease.(5^(th) ed). Philadelphia, W. B. Saunders Company, 1994). Acutepancreatitis is an important clinical problem with significant morbidityand mortality (Banerjee et al., British Journal of Surgery 81:1096–1103(1994)). The pathogenesis of this disease is still somewhat unresolvedbut it is widely recognized that pancreatic enzymes are released withinthe pancreas leading to proteolysis, interstitial inflammation, fatnecrosis, and hemorrhage. Acute pancreatitis can lead to disseminatedintravascular coagulation, adult respiratory distress syndrome, shock,and acute renal tubular necrosis (Cotran et al. Pathologic basis ofdisease. (5^(th) ed). Philadelphia, W. B. Saunders Company, 1994).Despite palliative measures, about 5% of these patients die of shockduring the first week of the clinical course. In surviving patients,sequelae may include pancreatic abscess, pseudocyst, and duodenalobstruction (Cotran et al. Pathologic basis of disease. (5^(th) ed).Philadelphia, W.B. Saunders Company, 1994). Chronic pancreatitis isoften a progressive destruction of the pancreas caused by repeatedflare-ups of acute pancreatitis. Chronic pancreatitis appears to incur amodestly increased risk of pancreatic carcinoma (Cotran et al.Pathologic basis of disease. (5^(th) ed). Philadelphia, W.B. SaundersCompany, 1994).

As indicated above and in Example 31, KGF-2 also promotes proliferationof pancreatic cells. Thus, in a further aspect, KGF-2 can be usedprophylactically or therapeutically to prevent or attenuate acute orchronic pancreatitis.

KGF-2 can also be used to reduce the side effects of gut toxicity thatresult from the treatment of viral infections, radiation therapy,chemotherapy or other treatments. KGF-2 may have a cytoprotective effecton the small intestine mucosa. KGF-2 may also be used prophylacticallyor therapeutically to prevent or attenuate mucositis and to stimulatehealing of mucositis (e.g., oral, esophageal, intestinal, colonic,rectal, and anal ulcers) that result from chemotherapy, other agents andviral infections. Thus the present invention also provides a method forpreventing or treating diseases or pathological events of the mucosa,including ulcerative colitis, Crohn's disease, and other diseases wherethe mucosa is damaged, comprising the administration of an effectiveamount of KGF-2. The present invention similarly provides a method forpreventing or treating oral (including odynophagia associated withmucosal injury in the pharynx and hypopharynx), esophageal, gastric,intestinal, colonic and rectal mucositis irrespective of the agent ormodality causing this damage.

In addition, KGF-2 could be used to treat and/or prevent: blisters andburns due to chemicals; ovary injury, for example, due to treatment withchemotherapeutics or treatment with cyclophosphamide; radiation- orchemotherapy-induced cystitis; or high-dose chemotherapy-inducedintestinal injury. KGF-2 could be used to promote internal healing,donor site healing, internal surgical wound healing, or healing ofincisional wounds made during cosmetic surgery.

KGF-2 can promote proliferation of endothelial cells, keratinocytes, andbasal keratinocytes. Thus, the present invention also provides a methodfor stimulating the proliferation of such cell types which involvescontacting cells with an effective amount of KGF-2. KGF-2 may beadministered to an individual in an effective amount to stimulate cellproliferation in vivo or KGF-2 may be contacted with such cells invitro.

The present invention further provides a method for promoting urothelialhealing comprising administering an effective amount of KGF-2 to anindividual. Thus, the present invention provides a method foraccelerating the healing or treatment of a variety of pathologiesinvolving urothelial cells (i.e., cells which line the urinary tract).Tissue layers comprising such cells may be damaged by numerousmechanisms including catheterization, surgery, or bacterial infection(e.g., infection by an agent which causes a sexually transmitteddisease, such as gonorrhea).

The present invention also encompasses methods for the promotion oftissue healing in the female genital tract comprising the administrationof an effective amount of KGF-2. Tissue damage in the female genitaltract may be caused by a wide variety of conditions including Candidainfections trichomoniasis, Gardnerella, gonorrhea, chlamydia, mycoplasmainfections and other sexually transmitted diseases.

As shown in Examples 10, 18, and 19, KGF-2 stimulates the proliferationof epidermal keratinocytes and increases epidermal thickening. Thus,KGF-2 can be used in full regeneration of skin; in full and partialthickness skin defects, including burns (i.e., repopulation of hairfollicles, sweat glands, and sebaceous glands); and the treatment ofother skin defects such as psoriasis.

KGF-2 can be used to treat epidermolysis bullosa, a defect in adherenceof the epidermis to the underlying dermis which results in frequent,open and painful blisters by accelerating reepithelialization of theselesions. KGF-2 can also be used to treat gastric and duodenal ulcers andhelp heal the mucosal lining and regeneration of glandular mucosa andduodenal mucosal lining more rapidly. Inflammatory bowel diseases, suchas Crohn's disease and ulcerative colitis, are diseases which result indestruction of the mucosal surface of the small or large intestine,respectively. Thus, KGF-2 could be used to promote the resurfacing ofthe mucosal surface to aid more rapid healing and to prevent orattenuate progression of inflammatory bowel disease. KGF-2 treatment isexpected to have a significant effect on the production of mucusthroughout the gastrointestinal tract and could be used to protect theintestinal mucosa from injurious substances that are ingested orfollowing surgery. As noted above, KGF-2 can also be used to promotehealing of intestinal or colonic anastomosis. KGF-2 can further be usedto treat diseases associate with the under expression of KGF-2.

As shown in Example 32 below, KGF-2 stimulates proliferation of lungepithelial cells. Thus, KGF-2 can be administered prophylactically toreduce or prevent damage to the lungs caused by various pathologicalstates. KGF-2 can also be administered during or after a damaging eventoccurs to promote healing. For example, KGF-2 can stimulateproliferation and differentiation and promote the repair of alveoli andbronchiolar epithelium to prevent, attenuate, or treat acute or chroniclung damage. Emphysema, which results in the progressive loss ofalveoli, and inhalation injuries, i.e., resulting from smoke inhalationand burns, that cause necrosis of the bronchiolar epithelium and alveolicould be effectively treated using KGF-2 as could damage attributable tochemotherapy, radiation treatment, lung cancer, asthma, black lung andother lung damaging conditions. Also, KGF-2 could be used to stimulatethe proliferation of and differentiation of type II pneumocytes, whichmay help treat or prevent disease such as hyaline membrane diseases,such as infant respiratory distress syndrome and bronchopulmonarydysplasia, in premature infants.

The three causes of acute renal failure are prerenal (e.g., heartfailure), intrinsic (e.g., nephrotoxicity induced by chemotherapeuticagents) and postrenal (e.g., urinary tract obstruction) which lead torenal tubular cell death, obstruction of the tubular lumens, and backflow of filtrate into the glomeruli (reviewed by Thadhani et al. N.Engl. J. Med. 334:1448–1460 (1996)). Growth factors such as insulin-likegrowth factor I, osteogenic protein-1, hepatocyte growth factor, andepidermal growth factor have shown potential for ameliorating renaldisease in animal models. Taub et al. Cytokine 5:175–179 (1993);Vukicevic et al. J. Am. Soc. Nephrol. 7:1867 (1996). As shown in Example31 below, KGF-2 stimulates proliferation of renal epithelial cells and,thus, is useful for alleviating or treating renal diseases andpathologies such as acute and chronic renal failure and end stage renaldisease.

KGF-2 could stimulate the proliferation and differentiation of breasttissue and therefor could be used to promote healing of breast tissueinjury due to surgery, trauma, or cancer.

In addition, KGF-2 could be used treat or prevent the onset of diabetesmellitus. In patients with newly diagnosed Types I and II diabetes,where some islet cell function remains, KGF-2 could be used to maintainthe islet function so as to alleviate, delay or prevent permanentmanifestation of the disease. Also, KGF-2 could be used as an auxiliaryin islet cell transplantation to improve or promote islet cell function.

Further, the anti-inflammatory property of KGF-2, could be beneficialfor treating acute and chronic conditions in which inflammation is a keypathogenesis of the diseases including, but not limiting to, psoriasis,eczema, dermatitis and/or arthritis. Thus, the present inventionprovides a method for preventing or attenuating inflammation, anddiseases involving inflammation, in an individual comprising theadministration of an effective amount of KGF-2.

KGF-2 can be used to promote healing and alleviate damage of braintissue due to injury from trauma, surgery or chemicals.

In addition, since KGF-2 increases the thickness of the epidermis, theprotein could be used for improving aged skin, reducing wrinkles inskin, and reducing scarring after surgery. Scarring of wound tissuesoften involves hyperproliferation of dermal fibroblasts. As noted inExample 10, fibroblast proliferation is not stimulated by KGF-2.Therefore, KGF-2 appears to be mitogen specific for epidermalkeratinocytes and induces wound healing with minimal scarring. Thus, thepresent invention provides a method for promoting the healing of woundswith minimal scarring involving the administration of an effectiveamount of KGF-2 to an individual. KGF-2 may be administered prior to,during, and/or after the process which produces the wound (e.g.,cosmetic surgery, accidental or deliberate tissue trauma caused by asharp object).

As noted above, KGF-2 also stimulates the proliferation of keratinocytesand hair follicles and therefore can be used to promote hair growth frombalding scalp, and in hair transplant patients. Thus, the presentinvention further provides a method for promoting hair growth comprisingthe administration of an amount KGF-2 sufficient to stimulate theproduction of hair follicles.

The present invention also provides a method for protecting anindividual from the effects of ionizing radiation, chemotherapy, ortreatment with anti-viral agents comprising the administration of aneffective amount of KGF-2. The present invention further provides amethod for treating tissue damage which results from exposure toionizing radiation, chemotherapeutic agents, or anti-viral agentscomprising the administration of an effective amount of KGF-2. Anindividual may be exposed to ionizing radiation for a number of reasons,including for therapeutic purposes (e.g., for the treatment ofhyperproliferative disorders), as the result of an accidental release ofa radioactive isotope into the environment, or during non-invasivemedical diagnostic procedures (e.g., X-rays). Further, a substantialnumber of individuals are exposed to radioactive radon in their workplaces and homes. Long-term continuous environmental exposure has beenused to calculate estimates of lost life expectancy. Johnson, W. andKearfott, K., Health Phys. 73:312–319 (1997). As shown in Example 23,the proteins of the present invention enhance the survival of animalsexposed to radiation. Thus, KGF-2 can be used to increase survival rateof individuals suffering radiation-induced injuries, to protectindividuals from sub-lethal doses of radiation, and to increase thetherapeutic ratio of irradiation in the treatment of afflictions such ashyperproliferative disorders.

KGF-2 may also be used to protect individuals against dosages ofradiation, chemotherapeutic drugs or antiviral agents which normallywould not be tolerated. When used in this manner, or as otherwisedescribed herein, KGF-2 may be administered prior to, after, and/orduring radiation therapy/exposure, chemotherapy or treatment withanti-viral agents. High dosages of radiation and chemotherapeutic agentsmay be especially useful when treating an individual having an advancedstage of an affliction such as a hyperproliferative disorder.

In another aspect, the present invention provides a method forpreventing or treating conditions such as radiation-induced oral andgastro-intestinal injury, mucositis, intestinal fibrosis, proctitis,radiation-induced pulmonary fibrosis, radiation-induced pneumonitis,radiation-induced pleural retraction, radiation-induced hemopoieticsyndrome, radiation-induced myelotoxicity, comprising administering aneffective amount of KGF-2 to an individual.

KGF-2 may be used alone or in conjunction with one or more additionalagents which confer protection against radiation or other agents. Anumber of cytokines (e.g., IL-1, TNF, IL-6, IL-12) have been shown toconfer such protection. See, e.g., Neta, R. et al., J. Exp. Med.173:1177 (1991). Additionally, IL-11 has been shown to protect smallintestinal mucosal cells after combined irradiation and chemotherapy,Du, X. X. et al., Blood 83:33 (1994), and radiation-induced thoracicinjury. Redlich, C. A. et al., J. Immun. 157:1705–1710 (1996). Severalgrowth factors have also been shown to confer protection to radiationexposure, e.g., fibroblast growth factor and transforming growth factorbeta-3. Ding, I. et al., Acta Oncol. 36:337–340 (1997); Potten, C. etal., Br. J. Cancer 75:1454–1459 (1997).

Hemorrhagic cystitis is a syndrome associated with certain diseasestates as well as exposure to drugs, viruses, and toxins. It manifestsas diffuse bleeding of the endothelial lining of the bladder. Knowntreatments include intravesical, systemic, and nonpharmacologictherapies (West, N.J., Pharmacotherapy 17:696–706 (1997). Some cytotoxicagents used clinically have side effects resulting in the inhibition ofthe proliferation of the normal epithelial in the bladder, leading topotentially life-threatening ulceration and breakdown in the epitheliallining. For example, cyclophosphamide is a cytotoxic agent which isbiotransformed principally in the liver to active alkylating metabolitesby a mixed function microsomal oxidase system. These metabolitesinterfere with the growth of susceptible rapidly proliferating malignantcells. The mechanism of action is believed to involve cross-linking oftumor cell DNA (Physicians' Desk reference, 1997).

Cyclophosphamide is one example of a cytotoxic agent which causeshemorrhagic cystitis in some patients, a complication which can besevere and in some cases fatal. Fibrosis of the urinary bladder may alsodevelop with or without cystitis. This injury is thought to be caused bycyclophosphamide metabolites excreted in the urine. Hematuria caused bycyclophosphamide usually is present for several days, but may persist.In severe cases medical or surgical treatment is required. Instances ofsevere hemorrhagic cystitis result in discontinued cyclophosphamidetherapy. In addition, urinary bladder malignancies generally occurwithin two years of cyclophosphamide treatment and occurs in patientswho previously had hemorrhagic cystitis (CYTOXAN (cyclophosphamide)package insert). Cyclophosphamide has toxic effects on the prostate andmale reproductive systems. Cyclophosphamide treatment can result in thedevelopment of sterility, and result in some degree of testicularatrophy.

As shown in FIGS. 52 and 53, systemic administration of KGF-2 to anindividual stimulates proliferation of bladder and prostatic epithelialcells. Thus, in one aspect, the present invention provides a method ofstimulating proliferation of bladder epithelium and prostatic epithelialcells by administering to an individual an effective amount of a KGF-2polypeptide. More importantly, as FIGS. 54 and 55 demonstrate, KGF-2 canbe used to reduce damage caused by cytotoxic agents having side effectsresulting in the inhibition of bladder and prostate epithelial cellproliferation. To reduce such damage, KGF-2 can be administered eitherbefore, after, or during treatment with or exposure to the cytotoxicagent. Accordingly, in a further aspect, there is provided a method ofreducing damage caused by an inhibition of the normal proliferation ofepithelial cells of the bladder or prostate by administering to anindividual an effective amount of KGF-2. As indicated, inhibitors ofnormal proliferation of bladder or prostate epithelium include radiationtherapy (causing acute or chronic radiation damage) and cytotoxic agentssuch as chemotherapeutic or antineoplastic drugs including, but notlimited to, cyclophosphamide, busulfan, and ifosfamide. In a furtheraspect, KGF-2 is administered to reduce or prevent fibrosis andulceration of the urinary bladder. Preferably, KGF-2 is administered toreduce or prevent hemorrhagic cystitis. Suitable doses, formulations,and administration routes are described below.

As used herein, by “individual” is intended an animal, preferably amammal (such as apes, cows, horses, pigs, boars, sheep, rodents, goats,dogs, cats, chickens, monkeys, rabbits, ferrets, whales, and dolphins),and more preferably a human.

The signal sequence of KGF-2 encoding amino acids 1 through 35 or 36 maybe employed to identify secreted proteins in general by hybridizationand/or computational search algorithms.

The nucleotide sequence of KGF-2 could be employed to isolate 5′sequences by hybridization. Plasmids comprising the KGF-2 gene under thecontrol of its native promoter/enhancer sequences could then be used inin vitro studies aimed at the identification of endogenous cellular andviral transactivators of KGF-2 gene expression.

The KGF-2 protein may also be employed as a positive control inexperiments designed to identify peptido-mimetics acting upon the KGF-2receptor.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA, manufacture of DNAvectors and for the purpose of providing diagnostics and therapeuticsfor the treatment of human disease.

Fragments of the full length KGF-2 gene may be used as a hybridizationprobe for a cDNA library to isolate the full length KGF-2 genes and toisolate other genes which have a high sequence similarity to these genesor similar biological activity. Probes of this type generally have atleast 20 bases. Preferably, however, the probes have at least 30 basesand generally do not exceed 50 bases, although they may have a greaternumber of bases. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete KGF-2 gene including regulatory and promotorregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the KGF-2 gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or cDNA to determinewhich members of the library the probe hybridizes to.

This invention provides a method for identification of the receptors forthe KGF-2 polypeptide. The gene encoding the receptor can be identifiedby numerous methods known to those of skill in the art, for example,ligand panning and FACS sorting (Coligan et al., Current Protocols inImmun., 1(2), Chapter 5 (1991)). Preferably, expression cloning isemployed wherein polyadenylated RNA is prepared from a cell responsiveto the polypeptides, and a cDNA library created from this RNA is dividedinto pools and used to transfect COS cells or other cells that are notresponsive to the polypeptides. Transfected cells which are grown onglass slides are exposed to the labeled polypeptides. The polypeptidescan be labeled by a variety of means including iodination or inclusionof a recognition site for a site-specific protein kinase. Followingfixation and incubation, the slides are subjected to autoradiographicanalysis. Positive pools are identified and sub-pools are prepared andre-transfected using an iterative sub-pooling and rescreening process,eventually yielding a single clone that encodes the putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to x-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

This invention provides a method of screening compounds to identifythose which agonize the action of KGF-2 or block the function of KGF-2.An example of such an assay comprises combining a mammalian Keratinocytecell, the compound to be screened and 3[H] thymidine under cell cultureconditions where the keratinocyte cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of keratinocyte proliferation in thepresence of the compound to determine if the compound stimulatesproliferation of Keratinocytes.

To screen for antagonists, the same assay may be prepared in thepresence of KGF-2 and the ability of the compound to preventKeratinocyte proliferation is measured and a determination of antagonistability is made. The amount of Keratinocyte cell proliferation ismeasured by liquid scintillation chromatography which measures theincorporation of ³[H] thymidine.

In another method, a mammalian cell or membrane preparation expressingthe KGF-2 receptor would be incubated with labeled KGF-2 in the presenceof the compound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of KGF-2 andreceptor would be measured and compared in the presence or absence ofthe compound. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

Examples of potential KGF-2 antagonists include an antibody, or in somecases, an oligonucleotide, which binds to the polypeptide.Alternatively, a potential KGF-2 antagonist may be a mutant form ofKGF-2 which binds to KGF-2 receptors, however, no second messengerresponse is elicited and therefore the action of KGF-2 is effectivelyblocked.

Another potential KGF-2 antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5 coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al.,Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)),thereby preventing transcription and the production of KGF-2. Theantisense RNA oligonucleotide hybridizes to the cDNA in vivo and blockstranslation of the cDNA molecule into KGF-2 polypeptide(Antisense—Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of KGF-2.

Potential KGF-2 antagonists include small molecules which bind to andoccupy the binding site of the KGF-2 receptor thereby making thereceptor inaccessible to KGF-2 such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall peptides or peptide-like molecules.

The KGF-2 antagonists may be employed to prevent the induction of newblood vessel growth or angiogenesis in tumors. Angiogenesis stimulatedby KGF-2 also contributes to several pathologies which may also betreated by the antagonists of the present invention, including diabeticretinopathy, and inhibition of the growth of pathological tissues, suchas in rheumatoid arthritis.

KGF-2 antagonists may also be employed to treat glomerulonephritis,which is characterized by the marked proliferation of glomerularepithelial cells which form a cellular mass filling Bowman's space.

The antagonists may also be employed to inhibit the over-production ofscar tissue seen in keloid formation after surgery, fibrosis aftermyocardial infarction or fibrotic lesions associated with pulmonaryfibrosis and restenosis. KGF-2 antagonists may also be employed to treatother proliferative diseases which are stimulated by KGF-2, includingcancer and Kaposi's sarcoma.

KGF-2 antagonists may also be employed to treat keratitis which is achronic infiltration of the deep layers of the cornea with uvealinflammation characterized by epithelial cell proliferation.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The polypeptides, agonists and antagonists of the present invention maybe employed in combination with a suitable pharmaceutical carrier tocomprise a pharmaceutical composition. Such compositions comprise atherapeutically effective amount of the polypeptide, agonist orantagonist and a pharmaceutically acceptable carrier or excipient. Sucha carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainers can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides, agonists and antagonists of the present invention may beemployed in conjunction with other therapeutic compounds.

The polypeptide having KGF-2 activity may be administered inpharmaceutical compositions in combination with one or morepharmaceutically acceptable excipients. It will be understood that, whenadministered to a human patient, the total daily usage of thepharmaceutical compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the type and degree ofthe response to be achieved; the specific composition an other agent, ifany, employed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the composition; the duration of the treatment; drugs(such as a chemotherapeutic agent) used in combination or coincidentalwith the specific composition; and like factors well known in themedical arts. Suitable formulations, known in the art, can be found inRemington's Pharmaceutical Sciences (latest edition), Mack PublishingCompany, Easton, Pa.

The KGF-2 composition to be used in the therapy will be formulated anddosed in a fashion consistent with good medical practice, taking intoaccount the clinical condition of the individual patient (especially theside effects of treatment with KGF-2 alone), the site of delivery of theKGF-2 composition, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The “effectiveamount” of KGF-2 for purposes herein is thus determined by suchconsiderations.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, intraarticular, subcutaneous, intranasal, intratrachealor intradermal routes. The pharmaceutical compositions are administeredin an amount which is effective for treating and/or prophylaxis of thespecific indication. In most cases, the dosage is from about 1 μg/kg toabout 30 mg/kg body weight daily, taking into account the routes ofadministration, symptoms, etc. However, the dosage can be as low as0.001 μg/kg. For example, in the specific case of topical administrationdosages are preferably administered from about 0.01 μg to 9 mg per cm².

As a general proposition, the total pharmaceutically effective amount ofthe KGF-2 administered parenterally per more preferably dose will be inthe range of about 1 μg/kg/day to 100 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. If given continuously, the KGF-2 is typically administeredat a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by1–4 injections per day or by continuous subcutaneous infusions, forexample, using a mini-pump. An intravenous bag solution or bottlesolution may also be employed.

A course of KGF-2 treatment to affect the fibrinolytic system appears tobe optimal if continued longer than a certain minimum number of days, 7days in the case of the mice. The length of treatment needed to observechanges and the interval following treatment for responses to occurappears to vary depending on the desired effect. Such treatment lengthsare indicated in the Examples below.

The KGF-2 polypeptide is also suitably administered by sustained-releasesystems. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers 22:547–556(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.Biomed. Mater. Res. 15:167–277 (1981), and R. Langer, Chem. Tech.12:98–105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release KGF-2compositions also include liposomally entrapped KGF-2. Liposomescontaining KGF-2 are prepared by methods known per se: DE 3,218,121;Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688–3692 (1985); Hwanget al., Proc. Natl. Acad. Sci. USA 77:4030–4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl.83–118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200–800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal KGF-2 therapy.

For parenteral administration, in one embodiment, the KGF-2 isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the KGF-2uniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes. Suitable formulations, known in the art, can be found inRemington's Pharmaceutical Sciences (latest edition), Mack PublishingCompany, Easton, Pa.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

KGF-2 is typically formulated in such vehicles at a concentration ofabout 0.01 μg/ml to 100 mg/ml, preferably 0.01 μg/ml to10 mg/ml, at a pHof about 3 to 8. It will be understood that the use of certain of theforegoing excipients, carriers, or stabilizers will result in theformation of KGF-2 salts.

KGF-2 to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic KGF-2compositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

KGF-2 ordinarily will be stored in unit or multi-dose containers, forexample, sealed ampules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous KGF-2 solution, and the resultingmixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized KGF-2 using bacteriostaticWater-for-Injection.

Dosaging may also be arranged in a patient specific manner to provide apredetermined concentration of an KGF-2 activity in the blood, asdetermined by an RIA technique, for instance. Thus patient dosaging maybe adjusted to achieve regular on-going trough blood levels, as measuredby RIA, on the order of from 50 to 1000 ng/ml, preferably 150 to 500ng/ml.

Pharmaceutical compositions of the invention may be administered orally,rectally, parenterally, intracisternally, intradermally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, creams,drops or transdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Preferred KGF-2 formulations are described in U.S. Provisional Appln.No. 60/068,493, filed Dec. 22, 1997, which is herein incorporated byreference.

The KGF-2 polypeptides, agonists and antagonists which are polypeptidesmay also be employed in accordance with the present invention byexpression of such polypeptides in vivo, which is often referred to as“gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.Further, before the cells are reintroduced into the patient, they may beseeded onto cell carriers, including biodegradable matrices (e.g.polyglycolic acid), tissue substitutes or equivalents (ex. artificialskin), artificial organs, and collagen derived matrices, etc.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle. Examples of other delivery vehicles include an HSV-based vectorsystem, adeno-associated virus vectors, and inert vehicles, for example,dextran coated ferrite particles.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller et al., Biotechniques Vol. 7, No. 9:980–990 (1989), or any otherpromoter (e.g., cellular promoters such as eukaryotic cellular promotersincluding, but not limited to, the histone, pol III, and β-actinpromoters). Other viral promoters which may be employed include, but arenot limited to, adenovirus promoters, thymidine kinase (TK) promoters,and B19 parvovirus promoters. The selection of a suitable promoter willbe apparent to those skilled in the art from the teachings containedherein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cell lineswhich may be transfected include, but are not limited to, the PE501,PA317, ψ-2, ψ-AM, PA12, T19-14×, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy 1:5–14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429–4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes.

The compounds or compositions may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceutical compoundsor compositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527–1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp.353–365 (1989); Lopez-Berestein, ibid., pp. 317–327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115–138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527–1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864–1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Antibody-Based Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10^(—2) M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁴M, 10⁴M, 5×10⁻⁵M, 10⁻⁵M, 5×10⁻⁶M, 10⁻⁶M,5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹¹M, 10⁻¹² M, 5×10⁻¹³M, 10⁻¹³ M, 5×10⁻¹⁴M,10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

Chromosome Assays

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15–25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 50 or 60bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D., et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

A cell has been “transformed” by exogenous DNA when such exogenous DNAhas been introduced inside the cell membrane. Exogenous DNA may or maynot be integrated (covalently linked) inter-chromosomal DNA making thegenome of the cell. Prokaryote and yeast, for example, the exogenous DNAmay be maintained on an episomal element, such a plasmid. With respectto eukaryotic cells, a stably transformed or transfected cell is one inwhich the exogenous DNA has become integrated into the chromosome sothat it is inherited by daughter cells through chromosome replication.This ability is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercell containing the exogenous DNA. An example of transformation isexhibited in Graham, F. & Van der Eb, A., Virology, 52:456–457 (1973).

“Transduction” or “transduced” refers to a process by which cells takeup foreign DNA and integrate that foreign DNA into their chromosome.Transduction can be accomplished, for example, by transfection, whichrefers to various techniques by which cells take up DNA, or infection,by which viruses are used to transfer DNA into cells.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating disorders, diseases and conditions. The gene therapy methodsrelate to the introduction of nucleic acid (DNA, RNA and antisense DNAor RNA) sequences into an animal to achieve expression of the KGF-2polypeptide of the present invention. This method requires apolynucleotide which codes for a KGF-2 polypeptide operatively linked toa promoter and any other genetic elements necessary for the expressionof the polypeptide by the target tissue. Such gene therapy and deliverytechniques are known in the art, see, for example, WO90/11092, which isherein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to aKGF-2 polynucleotide ex vivo, with the engineered cells then beingprovided to a patient to be treated with the polypeptide. Such methodsare well-known in the art. For example, see Belldegrun, A., et al., J.Natl. Cancer Inst. 85: 207–216 (1993); Ferrantini, M. et al., CancerResearch 53:1107–1112 (1993); Ferrantini, M. et al., J. Immunology153:4604–4615 (1994); Kaido, T. et al., Int. J. Cancer 60:221–229(1995); Ogura, H. et al., Cancer Research 50:5102–5106 (1990);Santodonato, L. et al., Human Gene Therapy 7:1–10 (1996); Santodonato,L. et al., Gene Therapy 4:1246–1255 (1997); and Zhang, J.-F. et al.,Cancer Gene Therapy 3:31–38 (1996)), which are herein incorporated byreference. In one embodiment, the cells which are engineered arearterial cells. The arterial cells may be reintroduced into the patientthrough direct injection to the artery, the tissues surrounding theartery, or through catheter injection.

As discussed in more detail below, the KGF-2 polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). The KGF-2polynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

In one embodiment, the KGF-2 polynucleotide is delivered as a nakedpolynucleotide. The term “naked” polynucleotide, DNA or RNA refers tosequences that are free from any delivery vehicle that acts to assist,promote or facilitate entry into the cell, including viral sequences,viral particles, liposome formulations, lipofectin or precipitatingagents and the like. However, the KGF-2 polynucleotides can also bedelivered in liposome formulations and lipofectin formulations and thelike can be prepared by methods well known to those skilled in the art.Such methods are described, for example, in U.S. Pat. Nos. 5,593,972,5,589,466, and 5,580,859, which are herein incorporated by reference.

The KGF-2 polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication.Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; pSVK3, pBPV, pMSG and pSVL available fromPharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of KGF-2 DNA. Suitable promoters includeadenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs; the b-actin promoter; and human growthhormone promoters. The promoter also may be the native promoter forKGF-2.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The KGF-2 polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked acid sequence injection, an effective dosage amount of DNAor RNA will be in the range of from about 0.05 mg/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked KGF-2 DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

As is evidenced in the Examples, naked KGF-2 nucleic acid sequences canbe administered in vivo results in the successful expression of KGF-2polypeptide in the femoral arteries of rabbits.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the KGF-2 polynucleotide constructs arecomplexed in a liposome preparation. Liposomal preparations for use inthe instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413–7416, which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077–6081, which isherein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem. (1990) 265:10189–10192, which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl. Acad. Sci. USA (1987) 84:7413–7416, which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413–7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512–527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ratio will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication no. WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are be engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding KGF-2. Retroviruses from which the retroviral plasmid vectorsmay be derived include, but are not limited to, Moloney Murine LeukemiaVirus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus,avian leukosis virus, gibbon ape leukemia virus, human immunodeficiencyvirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5–14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding KGF-2. Such retroviral vectorparticles then may be employed, to transduce eukaryotic cells, either invitro or in vivo. The transduced eukaryotic cells will express KGF-2.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with KGF-2 polynucleotide contained in an adenovirus vector. Adenoviruscan be manipulated such that it encodes and expresses KGF-2, and at thesame time is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. Adenovirus expression is achieved withoutintegration of the viral DNA into the host cell chromosome, therebyalleviating concerns about insertional mutagenesis. Furthermore,adenoviruses have been used as live enteric vaccines for many years withan excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev.Respir. Dis.109:233–238). Finally, adenovirus mediated gene transfer hasbeen demonstrated in a number of instances including transfer ofalpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431–434; Rosenfeld et al., (1992) Cell68:143–155). Furthermore, extensive studies to attempt to establishadenovirus as a causative agent in human cancer were uniformly negative(Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499–503 (1993); Rosenfeld et al., Cell 68:143–155 (1992);Engelhardt et al., Human Genet. Ther. 4:759–769 (1993); Yang et al.,Nature Genet. 7:362–369 (1994); Wilson et al., Nature 365:691–692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, for example, the HARP promoter of the present invention, butcannot replicate in most cells. Replication deficient adenoviruses maybe deleted in one or more of all or a portion of the following genes:E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The KGF-2 polynucleotideconstruct is inserted into the AAV vector using standard cloningmethods, such as those found in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAVvector is then transfected into packaging cells which are infected witha helper virus, using any standard technique, including lipofection,electroporation, calcium phosphate precipitation, etc. Appropriatehelper viruses include adenoviruses, cytomegaloviruses, vacciniaviruses, or herpes viruses. Once the packaging cells are transfected andinfected, they will produce infectious AAV viral particles which containthe KGF-2 polynucleotide construct. These viral particles are then usedto transduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the KGF-2 polynucleotide construct integrated intoits genome, and will express KGF-2.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding KGF-2) via homologous recombination (see, e.g., U.S. Pat.No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932–8935 (1989); and Zijlstra et al., Nature 342:435–438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not normally expressed in the cells, or isexpressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the KGF-2 desired endogenous polynucleotide sequenceso the promoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous KGF-2 sequence is placedunder the control of the promoter. The promoter then drives theexpression of the endogenous KGF-2 sequence.

The polynucleotides encoding KGF-2 may be administered along with otherpolynucleotides encoding other angiogenic proteins. Angiogenic proteinsinclude, but are not limited to, acidic and basic fibroblast growthfactors, VEGF-1, epidermal growth factor alpha and beta,platelet-derived endothelial cell growth factor, platelet-derived growthfactor, tumor necrosis factor alpha, hepatocyte growth factor, insulinlike growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

Preferably, the polynucleotide encoding KGF-2 contains a secretorysignal sequence that facilitates secretion of the protein. Typically,the signal sequence is positioned in the coding region of thepolynucleotide to be expressed towards or at the 5′ end of the codingregion. The signal sequence may be homologous or heterologous to thepolynucleotide of interest and may be homologous or heterologous to thecells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers (Kaneda et al.,Science 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277–11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits, sheep, cattle, horses and pigs,with humans being particularly preferred.

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488–505 (1993); Wu and Wu, Biotherapy 3:87–95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573–596 (1993);Mulligan, Science 260:926–932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191–217 (1993); May, TIBTECH 11(5): 155–215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932–8935 (1989); Zijlstra et al., Nature 342:435–438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429–4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932–8935 (1989); Zijlstra et al., Nature342:435–438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581–599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291–302(1994), which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644–651 (1994); Kiem et al., Blood 83:1467–1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129–141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110–114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499–503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3–10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431–434 (1991); Rosenfeld et al., Cell 68:143–155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225–234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775–783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289–300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599–618 (1993); Cohen et al., Meth. Enzymol. 217:618–644 (1993);Cline, Pharmac. Ther. 29:69–92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asT-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g., PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973–985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription. Demonstration of Therapeutic or Prophylactic Activity.

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Immune Activity

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, may be useful in treating deficiencies or disorders of the immunesystem, by activating or inhibiting the proliferation, differentiation,or mobilization (chemotaxis) of immune cells. Immune cells developthrough a process called hematopoiesis, producing myeloid (platelets,red blood cells, neutrophils, and macrophages) and lymphoid (B and Tlymphocytes) cells from pluripotent stem cells. The etiology of theseimmune deficiencies or disorders may be genetic, somatic, such as canceror some autoimmune disorders, acquired (e.g., by chemotherapy ortoxins), or infectious. Moreover, KGF-2 polynucleotides or polypeptides,or agonists or antagonists of KGF-2, can be used as a marker or detectorof a particular immune system disease or disorder.

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, may be useful in treating or detecting deficiencies or disordersof hematopoietic cells. KGF-2 polynucleotides or polypeptides, oragonists or antagonists of KGF-2, could be used to increasedifferentiation and proliferation of hematopoietic cells, including thepluripotent stem cells, in an effort to treat those disorders associatedwith a decrease in certain (or many) types of hematopoietic cells.Examples of immunologic deficiency syndromes include, but are notlimited to: blood protein disorders (e.g. agammaglobulinemia,dysgammaglobulinemia), ataxia telangiectasia, common variableimmunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection,leukocyte adhesion deficiency syndrome, lymphopenia, phagocytebactericidal dysfunction, severe combined immunodeficiency (SCIDs),Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

Moreover, KGF-2 polynucleotides or polypeptides, or agonists orantagonists of KGF-2, can also be used to modulate hemostatic (thestopping of bleeding) or thrombolytic activity (clot formation). Forexample, by increasing hemostatic or thrombolytic activity, KGF-2polynucleotides or polypeptides, or agonists or antagonists of KGF-2,could be used to treat blood coagulation disorders (e.g.,afibrinogenemia, factor deficiencies), blood platelet disorders (e.g.thrombocytopenia), or wounds resulting from trauma, surgery, or othercauses. Alternatively, KGF-2 polynucleotides or polypeptides, oragonists or antagonists of KGF-2, that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting,important in the treatment of heart attacks (infarction), strokes, orscarring.

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, may also be useful in treating or detecting autoimmune disorders.Many autoimmune disorders result from inappropriate recognition of selfas foreign material by immune cells. This inappropriate recognitionresults in an immune response leading to the destruction of the hosttissue. Therefore, the administration of KGF-2 polynucleotides orpolypeptides, or agonists or antagonists of KGF-2, that can inhibit animmune response, particularly the proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventingautoimmune disorders.

Examples of autoimmune disorders that can be treated or detectedinclude, but are not limited to: Addison's Disease, hemolytic anemia,antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergicencephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves'Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia,Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter'sDisease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic LupusErythematosus, Autoimmune Pulmonary Inflammation, Guillain-BarreSyndrome, insulin dependent diabetes mellitis, and autoimmuneinflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by KGF-2 polynucleotides or polypeptides, or agonists orantagonists of KGF-2. Moreover, these molecules can be used to treatanaphylaxis, hypersensitivity to an antigenic molecule, or blood groupincompatibility.

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, may also be used to treat and/or prevent organ rejection orgraft-versus-host disease (GVHD). Organ rejection occurs by host immunecell destruction of the transplanted tissue through an immune response.Similarly, an immune response is also involved in GVHD, but, in thiscase, the foreign transplanted immune cells destroy the host tissues.The administration of KGF-2 polynucleotides or polypeptides, or agonistsor antagonists of KGF-2, that inhibits an immune response, particularlythe proliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing organ rejection or GVHD.

Similarly, KGF-2 polynucleotides or polypeptides, or agonists orantagonists of KGF-2, may also be used to modulate inflammation. Forexample, KGF-2 polynucleotides or polypeptides, or agonists orantagonists of KGF-2, may inhibit the proliferation and differentiationof cells involved in an inflammatory response. These molecules can beused to treat inflammatory conditions, both chronic and acuteconditions, including inflammation associated with infection (e.g.,septic shock, sepsis, or systemic inflammatory response syndrome(SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, can be used to treat or detect hyperproliferative disorders,including neoplasms. KGF-2 polynucleotides or polypeptides, or agonistsor antagonists of KGF-2, may inhibit the proliferation of the disorderthrough direct or indirect interactions. Alternatively, KGF-2polynucleotides or polypeptides, or agonists or antagonists of KGF-2,may proliferate other cells which can inhibit the hyperproliferativedisorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated or detectedby KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, include, but are not limited to neoplasms located in the:abdomen, bone, breast, digestive system, liver, pancreas, peritoneum,endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary,thymus, thyroid), eye, head and neck, nervous (central and peripheral),lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, andurogenital.

Similarly, other hyperproliferative disorders can also be treated ordetected by KGF-2 polynucleotides or polypeptides, or agonists orantagonists of KGF-2. Examples of such hyperproliferative disordersinclude, but are not limited to: hypergammaglobulinemia,lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis,Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease,histiocytosis, and any other hyperproliferative disease, besidesneoplasia, located in an organ system listed above.

Cardiovascular Disorders

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, encoding KGF-2 may be used to treat cardiovascular disorders,including peripheral artery disease, such as limb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such asarterio-arterial fistula, arteriovenous fistula, cerebral arteriovenousmalformations, congenital heart defects, pulmonary atresia, and ScimitarSyndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's Syndrome, trilogy of Fallot, and ventricular heart septaldefects.

Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebralamyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arteriovenous malformation, cerebral arterydiseases, cerebral embolism and thrombosis, carotid artery thrombosis,sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epiduralhematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebralinfarction, cerebral ischemia (including transient), subclavian stealsyndrome, periventricular leukomalacia, vascular headache, clusterheadache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, are especially effective for the treatment of critical limbischemia and coronary disease. As shown in the Examples, administrationof KGF-2 polynucleotides and polypeptides to an experimentally inducedischemia rabbit hindlimb may restore blood pressure ratio, blood flow,angiographic score, and capillary density.

KGF-2 polypeptides may be administered using any method known in theart, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, biolistic injectors, particle accelerators, gelfoam spongedepots, other commercially available depot materials, osmotic pumps,oral or suppositorial solid pharmaceutical formulations, decanting ortopical applications during surgery, aerosol delivery. Such methods areknown in the art. KGF-2 polypeptides may be administered as part of apharmaceutical composition, described in more detail below. Methods ofdelivering KGF-2 polynucleotides are described in more detail herein.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345–355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630–634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757–1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401–411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175–203 (1985);Patz, Am. J. Opthalmol. 94:715–743 (1982); and Folkman et al., Science221:719–725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442–447 (1987).

The present invention provides for treatment of diseases or disordersassociated with neovascularization by administration of the KGF-2polynucleotides and/or polypeptides of the invention, as well asagonists or antagonists of KGF-2. Malignant and metastatic conditionswhich can be treated with the polynucleotides and polypeptides, oragonists or antagonists of the invention include, but are not limitedto, malignancies, solid tumors, and cancers described herein andotherwise known in the art (for a review of such disorders, see Fishmanet al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).

Ocular disorders associated with neovascularization which can be treatedwith the KGF-2 polynucleotides and polypeptides of the present invention(including KGF-2 agonists and/or antagonists) include, but are notlimited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma,retrolental fibroplasia, uveitis, retinopathy of prematurity maculardegeneration, corneal graft neovascularization, as well as other eyeinflammatory diseases, ocular tumors and diseases associated withchoroidal or iris neovascularization. See, e.g., reviews by Waltman etal., Am. J. Ophthal. 85:704–710 (1978) and Gartner et al., Surv.Ophthal. 22:291–312 (1978).

Additionally, disorders which can be treated with the KGF-2polynucleotides and polypeptides of the present invention (includingKGF-2 agonist and/or antagonists) include, but are not limited to,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,delayed wound healing, granulations, hemophilic joints, hypertrophicscars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

Moreover, disorders and/or states, which can be treated with the KGF-2polynucleotides and polypeptides of the present invention (includingKGF-2 agonist and/or antagonists) include, but are not limited to, solidtumors, blood born tumors such as leukemias, tumor metastasis, Kaposi'ssarcoma, benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis,psoriasis, ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis, retinoblastoma, and uvietis, delayed wound healing,endometriosis, vascluogenesis, granulations, hypertrophic scars(keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

Digestive Diseases

KGF-2 has been shown to stimulate the proliferation of cells of thegastrointestinal tract. Thus, KGF-2 polynucleotides, polypeptides,agonists, and/or antagonists can be used to treat and/or detectdigestive diseases.

Examples of digestive diseases which can be treated or detected include:biliary tract diseases (such as bile duct diseases which include bileduct neoplasms, bile duct obstruction, Caroli's disease, cholangitis;common bile duct diseases such as choledochal cyst, common bile ductcalculi, and common bile duct neoplasms; bile reflux, biliary atresia,biliary dyskinesia, biliary fistula, biliary tract neoplasms,gallbladder neoplasms, cholelithiasis such as common bile duct calculi;cholestasis, bile duct obstruction, alagille syndrome and livercirrhosis; gallbladder diseases such as cholecystitis, cholelithiasisand gallbladder neoplasms; hemobilia and postcholecystectomy syndrome),digestive system abnormalities (such as imperforate anus, Barrettesophagus, biliary atresia, diaphragmatic eventration, esophagealatresia, Hirschsprung Disease, intestinal atresia, Meckel'sDiverticulum), digestive system fistula (which includes biliary fistulaand esophageal fistula such as tracheoesophageal fistula, gastricfistula, intestinal fistula such as rectal fistula), digestive systemfistula (such as intestinal fistula such as rectal fistula whichincludes rectovaginal fistula and pancreatic fistula), digestive systemneoplasms (such as biliary tract neoplasms which includes common bileduct neoplasms, gallbladder neoplasms), esophageal neoplasms,gastrointestinal neoplasms, such as intestinal neoplasms such as cecalneoplasms which include appendiceal neoplasms such as colonic polypssuch as adenomatous polyposis coli, colorectal neoplasms such ashereditary colorectal neoplasms and nonpolyposis, sigmoid neoplasms,duodenal neoplasms, duodenal neoplasms, ileal neoplasms, intestinalpolyps such as colonic polyps such as adenomatous polyposis coli,Gardner Syndrome and Peutz-Jeghers Syndrome, jejunal neoplasms, rectalneoplasms such as anus neoplasms), digestive system neoplasms (such asgastrointestinal neoplasms such as intestinal neoplasms such as rectalneoplasms which include anus neoplasms and anal gland neoplasms, stomachneoplasms, pancreatic neoplasms and peritoneal neoplasms), esophagealdiseases (such as Barrett Esophagus, esophageal and gastric varices,esophageal atresia, esophageal cyst, esophageal diverticulum such asZenker's Diverticulum, esophageal motility disorders such as CRESTSyndrome, deglutition disorders such as Plummer-Vinson Syndrome,esophageal achalasia, diffuse esophageal spasm and gastroesophagealreflux, esophageal neoplasms, esophageal perforation such asMallory-Weiss Syndrome, esophageal stenosis, esophagitis such as pepticesophagitis, diaphragmatic hernia such as traumatic diaphragmatichernia, hiatal hernia.)

Examples of gastrointestinal diseases which can be treated or detectedinclude gastroenteritis such as cholera morbus, gastrointestinalhemorrhage (such as hematemesis, melena and peptic ulcer), hernia (suchas diaphragmatic hernia which include traumatic diaphragmatic hernia andhiatal hernia, femoral hernia, inguinal hernia, obturator hernia,umbilical hernia and ventral hernia), intestinal diseases (such as cecaldiseases which include appendicitis, cecal neoplasms such as appendicealneoplasms, colonic diseases such as colitis which include ischemiccolitis, ulcerative colitis such as toxic megacolon, enterocolitis suchas pseudomembranous entercolitis, proctocolitis, functional colonicdiseases such as colonic pseudo-obstruction, colonic neoplasms such ascolonic polyps such as adenomatous polyposis coli, colorectal neoplasmssuch as hereditary colorectal neoplasms and nonpolyposis, sigmoidneoplasms, colonic diverticulities, colonic diverticulosis, megacolonsuch as Hirschsprung Disease and toxic megacolon, sigmoid diseases suchas proctocolitis and sigmoid neoplasms, constipation, Crohn's disease,diarrhea such as infantile diarrhea, dysentery such as amebic dysenteryand bacillary dysentery, duodenal diseases such as duodenal neoplasms,duodenal obstruction such as superior mesenteric artery syndrome,duodenal ulcer such as Curling's Ulcer and duodenitis, enteritis such asenterocolitis which includes pseudomembranous entercolitis, ilealdiseases such as ileal neoplasms and ileitis, immunoproliferative smallintestinal disease, inflammatory bowel diseases such as ulcerativecolitis and Crohn's Disease, intestinal atresia, parasitic intestinaldiseases such as anisakiasis, balantidiasis, blastocystis infections,cryptosporidiosis, dientamoebiasis, dientamoebiasis, amebic dysenteryand giardiasis, intestinal fistula such as rectal fistula which includerectovaginal fistula, intestinal neoplasms such as cecal neoplasms whichinclude appendiceal neoplasms, colonic neoplasms such as colonic polypswhich include adenomatous polyposis coli, colorectal neoplasms such ashereditary colorectal neoplasms and nonpolyposis, sigmoid neoplasms,duodenal neoplasms, ileal neoplasms, intestinal polyps such as colonicpolyps such as adenomatous polyposis coli, Gardner Syndrome,Peutz-Jeghers Syndrome, intestinal obstruction such as afferent loopsyndrome, duodenal obstruction, impacted feces, intestinalpseudo-obstruction such as colonic pseudo-obstruction, intussusception,intestinal perforation, intestinal polyps such as colonic polyps whichinclude adenomatous polyposis coli, jejunal diseases such as jejunalneoplasms, malabsorption syndromes such as blind loop syndrome, celiacdisease, lactose intolerance, intestinal lipodystrophy, short bowelsyndrome, tropical sprue, occlusion mesenteric vascular, pneumatosiscystoides intestinalis, protein-losing enteropathies such as intestinallymphangiectasis, rectal diseases such as anus diseases which includeanus neoplasms such anal gland neoplasms, fissure in ano, pruritus ani,fecal incontinence, hemorrhoids, proctitis such as proctocolitis, rectalfistula such as rectovaginal fistula, rectal neoplasms such as anusneoplasms such as anal gland neoplasms, rectal diseases such as rectalprolapse, peptic ulcer, Peptic esophagitis, marginal ulcer, peptic ulcerhemorrhage, peptic ulcer perforation, stomach ulcer, Zollinger-EllisonSyndrome, postgastrectomy syndromes such as dumping syndrome, stomachdiseases such as achlorhydria, duodenogastric reflux such as bilereflux, gastric fistula, gastric mucosa prolapse, gastric outletobstruction such as pyloric stenosis, gastritis such as atrophicgastritis and hypertrophic gastritis, gastroparesis, stomach dilatation,stomach diverticulum, stomach neoplasms, stomach rupture, stomach ulcerand stomach volvulus, gastrointestinal tuberculosis, visceroptosis,vomiting such as hematemesis and hyperemesis gravidarum), pancreaticdiseases such as cystic fibrosis, pancreatic cyst such as pancreaticpseudocyst, pancreatic fistula, pancreatic insufficiency, pancreaticneoplasms and pancreatitis), peritoneal diseases such aschyloperitoneum, hemoperitoneum, mesenteric cyst, mesentericlymphadenitis, mesenteric vascular occlusion, peritoneal paniculitis,peritoneal neoplasms, peritonitis, pneumoperitoneum, subphrenic abscessand peritoneal tuberculosis.

Digestive diseases which may be treated or detected also include liverdiseases. Liver diseases include acute yellow atrophy, intrahepaticcholestasis such as alagille syndrome and biliary liver cirrhosis, fattyliver such as alcoholic fatty liver and Reye's Syndrome, hepatic veinthrombosis, hepatic veno-occlusive disease, hepatitis such as alcoholichepatitis, animal hepatitis such as animal viral hepatitis such asinfectious canine hepatitis and Rift Valley Fever, toxic hepatitis,human viral hepatitis such as delta infection, hepatitis A, hepatitis B,hepatitis C, chronic active hepatitis and hepatitis E, hepatolenticulardegeneration, hepatomegaly, hepatorenal syndrome, portal hypertensionsuch as Cruveilhier-Baumgarten Syndrome and Esophageal and gastricvarices, liver abscess such as amebic liver abscess, liver cirrhosissuch as alcoholic liver cirrhosis, biliary liver cirrhosis andexperimental liver cirrhosis, alcoholic liver diseases such as alcoholicfatty liver, alcoholic hepatitis and alcoholic liver cirrhosis,parasitic liver diseases such as hepatic echinococcosis, fascioliasis,and amebic liver abscess, liver failure such as hepatic encephalopathyand acute liver failure, liver neoplasms, peliosis hepatis,erythrohepatic porphyria, and hepatic porphyria such as acuteintermittent porphyria and porphyria cutanea tarda, hepatic tuberculosisand Zellweger Syndrome).

Examples of stomatognathic diseases which can be treated or detectedinclude jaw diseases (such as cherubism, giant cell granuloma, jawabnormalities such as cleft palate, micrognathism, Pierre RobinSyndrome, prognathism and retrognathism, jaw cysts such asnonodontogenic cysts, odontogenic cysts such as basal cell nevussyndrome, dentigerous cyst, calcifying odontogenic cyst, periodontalcyst such as radicular cyst, edentulous jaw such as partially edentulousjaw, jaw neoplasms such as mandibular neoplasms, maxillary neoplasms andpalatal neoplasms, mandibular diseases such as craniomandibulardisorders which include temporomandibular joint diseases such astemporomandibular joint syndrome, mandibular neoplasms, prognathism andretrognathism, maxillary diseases such as maxillary neoplasms), mouthdiseases (such as Behcet's Syndrome, Burning Mouth Syndrome, oralcandidiasis, dry socket, focal epithelial hyperplasia, oral leukoedema,oral lichen planus, lip diseases such as cheilitis, cleft lip, herpeslabialis and lip neoplasms, Ludwig's Angina, Melkersson-RosenthalSyndrome, mouth abnormalities such as cleft lip, cleft palate,fibromatosis gingivae, macroglossia, macrostomia, microstomia andvelopharyngeal insufficiency, edentulous mouth such as edentulous jawsuch as partially edentulous jaw, mouth neoplasms such as gingivalneoplasms such as gingival neoplasms, oral leukoplakia such as hairyleukoplakia, lip neoplasms, palatal neoplasms, salivary gland neoplasmssuch as parotid neoplasms, sublingual gland neoplasms and submandibulargland neoplasms and tongue neoplasms, noma, oral fistula such as dentalfistula, oroantral fistula and salivary gland fistula, oral hemorrhagesuch as gingival hemorrhage, oral manifestations, oral submucousfibrosis, periapical periodontitis such as periapical abscess andperiapical granuloma and radicular cyst), periodontal diseases (such asalveloar bone loss, furcation defects such as gingival hemorrhage,gingival hyperplasia, gingival hypertrophy, gingival neoplasms, gingivalrecession, gingivitis such as gingival crevicular fluid, gingivalpocket, necrotizing ulcerative gingivitis, giant cell granuloma andpericoronitis, periodontal attachment loss, periodontal cyst,periodontitis such as periodontal abscess, periodontal pocket andperiodontosis, tooth exfoliation, tooth loss, tooth migration such asmesial movement of teeth and tooth mobility), ranula, salivary glanddiseases (such as Mikulicz' Disease, parotid diseases such as parotidneoplasms and parotitis such as mumps, salivary gland calculi such assalivary duct calculi, salivary gland fistula, salivary gland neoplasmssuch as parotid neoplasms, sublingual gland neoplasms and submandibulargland neoplasms), sialadenitis, necrotizing sialometaplasia, sialorrhea,submandibular gland diseases such as submandibular gland neoplasms,xerostomia such as Sjogren's syndrome, stomatitis (such asStevens-Johnson Syndrome, aphthous stomatitis, aphthous stomatitis,denture stomatitis and herpetic stomatitis), tongue diseases (such asglossalgia, glossitis such as benign migratory glossitis), macroglossia,tongue diseases (such as fissured tongue, hairy tongue and tongueneoplasms and oral tuberculosis), pharyngeal diseases (such aspharyngeal diseases such as nasopharyngeal diseases such asnasopharyngeal neoplasms and nasopharyngitis), peritonsillar abscess,pharyngeal neoplasms such as hypopharyngeal neoplasms, nasopharyngealneoplasms and oropharyngeal neoplasms which include tonsillar neoplasms,pharyngitis, retropharyngeal abscess, tonsillitis and velopharyngealinsufficiency), stomatognathic system abnormalties, temporomandibularjoint diseases such as temporomandibular joint syndrome, tooth diseases(such as bruxism, dental depositis which includes dental calculus anddental plague, dental leakage, dental pulp diseases which includesdental pulp autolysis, dental pulp calcification, dental pulp exposure,dental pulp gangrene, secondary dentin and pulpitis, dentin sensitivity,dental focal infection, hypercementosis, malocclusion such as traumaticdental occlusion, diastema, angle class I malocclusion, angle class IImalocclusion, angle class III malocclusion, mottled enamel, toothabnormalities such as amelogenesis imperfecta such as dental enamelhypoplasia, anodonitia, dens in dente, dentin dysplasia, dentinogenesisimperfecta, fused teeth, odontodysplasia and supernumerary tooth, toothabrasion, tooth deminerlization such as dental caries which includesdental fissures and root caries, tooth discoloration, tooth erosion,ectopic tooth eruption, impacted tooth, tooth injuries such as toothFractures such as cracked tooth syndrome and tooth luxation, tooth loss,tooth resorption such as root resorption and unerupted tooth andtoothache).

Ocular Diseases

KGF-2 has been shown to stimulate proliferation of cells of the eye.Thus, KGF-2 polynucleotides, polypeptides, agonists, and/or antagonistscan be used to treat and/or detect ocular diseases.

Examples of ocular diseases which can be treated or detected includeasthenopia, conjunctival diseases, conjunctival neoplasms,conjunctivitis (allergic, bacterial, inclusion, ophthalmia neonatorum,trachoma, viral, acute hemorrhagic), keratoconjunctivitis,keratoconjunctivitis (infectious or sicca), Reiter's Disease, Pterygium,xerophthalmia, corneal diseases, corneal dystrophies (hereditary),Fuchs' Endothelial Dystrophy, corneal edema, corneal neovascularization,corneal opacity, arcus senilis, keratitis, acanthamoeba keratitis,corneal ulcer, herpetic keratitis, dendritic keratitis,keratoconjunctivitis, keratoconus, trachoma, eye abnormalities(aniridia, WAGR Syndrome, Anophthamos, blepharophimosis, coloboma,ectopia lentis, hydrophthalmos, microphthalmos, retinal dysplasia),hereditary eye diseases (albinism, ocular albinism, oculocutaneousalbinism, choroideremia, hereditary corneal dystrophies, gyrate atrophy,hereditary optic atrophy, retinal dysplasa, retinitis pigmentosa), eyehemorrhage (choroid hemorrhage, hyphema, retinal hemorrhage, vitreoushemmorrhage), eye infections (corneal ulcer, bacterial eye infections,bacterial conjunctivitis, inclusion conjunctivitis, ophthalmianeonatorum, trachoma, hordeolum, infectious keratoconjunctivitis, oculartuberculosis), fungal eye infections, parasitic eye infections(acanthamoeba keratitis, ocular onchocerciasis, ocular toxoplasmosis),viral eye infections (viral conjunctivitis, acute hemorrhagicconjunctivitis, cytomegalovirus retinitis, Herpes Zoster Ophthalmicus,herpetic keratitis, dendritic keratitis), suppurative uveitis(endophthalmitis, panophthalmitis), eye injuries (eye burns, eye foreignbodies, penetrating eye injuries), eye manifestations, eye neoplasms(conjunctival neoplasms, eyelid neoplasms, orbital neoplasms, uvealneoplasms (choroid neoplasms, iris neoplasms), eyelid diseases(blepharitis, blepharophimosis, blepharoptosis, belpharospasm,chalazion, ectropion, entropion, eyelid neoplasms, hordeolum), lacrimalaparatus diseases (dacroyocystitis, dry eye sundromes,keratoconjunctivitis sicca, Sjogren's Syndrome, xerophthalmia, lacrimalduct obstruction), lens diseases (aphakia, poscataract aphakia,cataract, lens subluxation, ectopia lentis, ocular hypertension,glaucoma (angle-closure, neovascular, open-angle, hydrophthalmos),ocular hypotension, ocular motility disorders (amblyopia, nystagmus,oculomotor nerve paralysis, ophthalmoplegia (Duane's Syndrome, Horner'sSyndrome, Chronic progressive external ophthalmoplegia, KearnsSyndrome), strabismus (esotropia), optic nerve diseases (optic atrophy,hereditary optic atrophy, optic disk drusen, optic neuritis,neuromyelitis optica, papilledema), orbital diseases (enophthalmos,exophthalmos, Graves' Disease, orbital plasma cell granuloma, orbitalneoplasms), abnomal pupillary functions (anisocoria, tonic pupil, Adie'sSyndrome, miosis, mydriasis, Homer's Syndrome), refractive errors(aniseikonia, anisometropia, astigmatism, hyperopia, myopia,presbyopia), retinal diseases (angioid streaks, diabetic retinopathy,retinal artery occusion, retinal degeneration, macular degeneration,cystoid macular edema, retinal drusen, retinitis pigmentosa, KearnsSyndrome, retinal detachment, retinal dysplasia, retinal hemorrhage,retinal neovascularization, retinal perforations, retinal veinocclusion, retinitis (chorioretinitis, cytomegalovirus retinitis, acuteretinal necrosis syndrome), retinopathy of prematurity, proliferativevitreoretinopathy), scleral diseases (scleritis), uveal diseases(choroid diseases, choroid hemorrahage, choroid neoplasms,choroideremia, choroiditis, chorioretinitis, pars lanitis, gyrateatrophy), iris diseases (exfoliation syndrome, iridocyclitis, irisneoplasms), uveitis (panuveitis, sympathetic ophthalmia, anteriorbehcet's syndrome, iriocyclitis, iritis, posterior uveitis, choroiditis,chorioretinitis, pars planitis, intermediate uveitis, pars planitis,suppurative uveitis (endophthalmitis, panophthalmitis),uveomeningoencephalitic syndrome), vision disorders (amblyoia,blindness, hemianopsia, color vision defects, diplopia, night blindness,scotoma, subnormal vision), and proliferative vitreoretinopathy.

Skin and Connective Tissue Diseases

KGF-2 stimulates the proliferation of the cells of the skin andconnective tissue. Therefore, KGF-2 polynucleotides, polypeptides,agonists, and/or antagonists can be used to treat and/or detect diseasesof the skin and/or connective tissue.

Examples of connective tissue diseases include: cartilage diseases, suchas relapsing polychondritis and Tietze's Syndrome; cellulitis; collagendiseases, such as Ehler's Danlos syndrome, keloids (including acnekeloids), mucopolysaddaridosis I, necrobiotic disorders (includinggranuloma annulare, necrobiosis lipoidica), and osteogenesis imperfecta;cutis laxa; dermatomyositis; Dupytren's contracture; homocystinuria;lupus erythematosis (including cutaneous, discoid, panniculitis,systemic and nephritis; marfan syndrome; mixed connective tissuedisease; mucinosis, including follicular, mucopolysaccaridoses (I, II,UU, IV, IV, and VII), myxedema, scleredemo adultorum and synovial cysts;connective tissue neoplasms; noonan syndromel osteopoikilosis;panniculitis, including erythema induratum, nodular nonsuppurative andperitoneal; penile induration; pseudoxanthoma elasticum; rheumaticdiseases, including arthritis (rheumatoid, juvenile rheumatoid, Caplan'ssyndrome, Felty's syndrome, rheumatoid nodule, ankylosing spondylitis,and still's disease), hyperostosis, polymyalgia rheumatics;circumscribed scleroderma, and systemic scleroderma (CREST syndrome).

Examples of skin diseases include angiolymphoid hyperplasia witheosinophilia; cicatix (including hypertophic); cutaneous fistula, cuislaxa; dermatitis, including acrodermatitis, atopic dermatitis, contactdermatitis (allergic contact, photoallergic, toxicodendron), irritantdermatitis (phototoxic, diaper rash), occupational dermatitits;exfoliative dermatitis, herpetiformis dermatittis, seborrheicdermatitis, drug eruptions (such as toxic epidermal necrolysis,eryuthema nodosum, serum sickness) eczema, including dyshidrotic,intertrigo, neurodermatitis, and radiodermatitis; dermatomyositis;erythema, including chronicum migrans, induratum, infectiosum,multiforme (Stevens-Johnson syndrome), and nodosum (Sweet's syndrome);exanthema, including subitum; facial dermatosis, including acneiformeruptions (keloid, rosacea, vulgaris and Favre-Racouchot syndrome); footdermatosis, including tinea pedis; hand dermatoses; keratoacanthoma;keratosis, inlcuding callosities, cholesteatoma (including middle ear),ichthyosis (including congentical ichtyosiform erythroderms,epidermolytic hyperkeratosis, lamellar ichthyosis, ichthyosis vulgaris,X-linked ichthyosis, and Sjogren-Larsson syndrome), keratodermablennorrhagicum, palmoplantar keratoderms, follicularis keratosis,seborrheic keratosis, parakeratosis and porokeratosis; leg dermatosis,mastocytosis (urticaria pigmentosa), necrobiotic disorders (granulomaannulare and necrobiosis lipoidica), photosensitivity disorders(photoallergic or photoxic dermatitis, hydroa vacciniforme, sundurn, andxeroderma pigmentosum); pigmentation disorders, including argyria,hyperpigmentation, melanosis, aconthosis nigricans, lentigo,Peutz-Jeghers syndrome, hypopigmentation, albinism, pibaldism, vitiglio,incontinentia pigmenti, urticaria pigmentosa, and xeroderma pigmentosum.

Further examples of skin disorders include prurigo; pruritis (includingani and vulvae); pyoderma, including ecthyma and pyoderma gangrenosum;sclap dermatoses; sclerodema adultorum; sclerma neonatorum; skinappenage diseases, including hair diseases (alopecia, folliculitis,hirsutism, hypertichosis, Kinky hair syndrome), nail diseases(nail-patella syndrome, ingrown or malformed nails, onychomycosis,paronychia), sebaceous gland diseases (rhinophyma, neoplasms), sweatgland diseases (hidradentitis, hyperhidrosis, hypohidrosis, miliara,Fox-Fordyce disease, neoplasms); genetic skin deseases, includingalfinism, cutis laxa, benign familial pemphigis, porphyria,acrodermatitis, ectodermal dysplasia, Ellis-Van Creveld syndrome, focaldermal hypoplasia, Ehlers-Danlos syndrome, epidermolysis bullosa,ichtysosis; infectious skin diseases, including dermatomycoses,blastomycosis, candidiasis, chromoblastomycosis, maduromycosis,paracoccidioidomycosis, sporotrichosis, tinea; bacterial skin diseases,such as cervicofacial actinomycosis, bacilliary angiomatosis, ecthyma,erysipelas, erythema chronicum migrans, erythrasma, granuloma inguinale,hidradenitis suppurativa, maduromycosis, paronychia, pinta,rhinoscleroma, staphylococcal skin infections (furuncolosis, carbuncle,impetigo, scalded skin syndrome), cutaneous syphilis, cutaneoustuberculosis, yaws; parasitic skin diseases, including larva migrans,Leishmaniasis, pediculosis, and scabies; viral skin diseases, includingeythema infectiosum, exanthema subitum, herpes simplex, moolusumcontagiosum, and warts.

Further examples of skin diseases include metabolic skin disesases, suchas adiposis dolorosa, lipodystrophy, necrobiosis lipoidica, porhphyria,juvenile xanthogranuloma, xanthomatosis (Wolman disease);papulosequamous skin diseases, including lichenoid eruptions,parpasoriasis, pityriasis, and psoriasis; vascular skin diseases, suchas Behcet's syndrome, mucocutaneous lymph node syndrome, polyarteritisnodosa, pyoderma gangernosum, Takayasu's arteritis; vesculobullous skindiseases, including acantholysis, blisters, herpes gestationis, hybroavacciniforme, pemphigoid, pemphigus; skin neoplasms; skin ulcers, suchas decubitus ulcer, leg ulcers, foot ulcers, diabetic foot ulcers,varicose ulcers and pyoderma gangrenosum.

Uro-Genital Diseases and Disorders

KGF-2 may stimulate the proliferation of the cells of the uro-genitaltract. Therefore, KGF-2 polynucleotides, polypeptides, agonists, and/orantagonists can be used to treat and/or detect male and female genitaldiseases and/or disorders and pregnancy complications.

Examples of urologic and male genital diseases which can be treated ordetected include epididymitis, male genital neoplasms, penile neoplasms,prostatic neoplasms, testicular neoplasms, hematocele, herpes genitalis,hydrocele, male infertility, oligospermia, penile diseases includingbalanitis, hypospadias, penile induration, penile neoplasms, phimosis,paraphimosis, priapism, prostatic diseases such as hypertrophy,neoplasms, and prostatitis, sexual disorders such as impotensce andvasculogenic impotence, spermatic cord torsion, spermatocele, testiculardiseases including cryptorchidism, orchitis, and testicular neoplasms,male genital tuberculosis, varicocele, urogenital tuberculosis (malegenital, renal), urogenital abnormalities, bladder exstrophy,cryptorchidism, epispadias, hypospadias, polycystic kidney (autosomaldominant and autosomal recessive), hereditary nephritis, sexdifferentiation disorders, gonadal dysgenesis, mixed gonadal dysgenesis,hermaphroditism, pseudohermaphroditism, Kallman Syndrome, Klinefelter'sSyndrome, testicular feminization, WAGR Syndrome, urogenital neoplasms,male genital neoplasms (penile, prostatic, testicular), urologicneoplasms (bladder, kidney, ureteral, urethral), bladder diseases(calculi, exstrophy, fistula, vesicovaginal fistula, neck obstruction,neoplasms, neurogenic, cystitis, vesico-ureteral reflux), hematuria,hemoglobinuria, AIDS-associated nephropathy, anuria, oliguria, diabeticnephropathies, Fanconi Syndrome, hepatorenal syndrome, hydronephrosis,primary hyperoxaluria, renal hypertension, renovascular hypertension,kidney calculi, kidney cortex necrosis, cystic kidney, polycystickidney, polycistic kidney (autosomal dominant, autosomal recessive),sponge kidney, kidney failure (nephrogenic disbetes insipidus, acutekidney failure, kidney papillary necrosis), nephritis(glomerulonephritis (IGA, membronoproliferative, membranous, focal,Goodpasture's Syndrome, Lupus Nephritis), hereditary nephritis,insterstitial nephritis, balkan nephropathy, pyelonephritis,xanthogranulomatous pyelonephritis, nephrocalcinosis, nephrosclerosis,nephrosis, lipoid nephrosis, nephrotic syndrome, perinephritis),pyelitis (pyelocystitis, pyelonephritis, xanthogranulomatouspyelonephritis), renal artery obstruction, renal osteodystrophy, inbornerrors in renal tubular transport, renal tubular acidosis, renalaminoaciduria, cystinuria, Hartnup Disease, Cystinosis, FranconiSyndrome, Renal glycosuria, familial hypophosphatemia, oculocerebrorenalsyndrome, psudohypoaldosteronism, renal tuberculosis, uremia,Hemolytic-Uremic Syndrome, Wegener's Granulomatosis, Zellweger Syndrome,proteinuria, albuminuria, ureteral diseases including ureteral calculi,ureteral neoplasms, ureteral obstructionm, ureterocele, urethraldiseases including epispadias, urethral neoplasms, urethralobstrauction, urethral stricture, urethritis (reiter's disease), urinarycalculi (bladder, kidney, ureteral), urinary fistula (bladder fistula(vesicovaginal fistula)), urinary tract infections (bacteruria, pyuria,schistosomiasis haematobia), and urination disorders (enuresis,polyuria, urinary incontinence, stress-related urinary incontinence,urinary retention).

Examples of female genital disease and pregnancy complications which canbe treated or detected include adnexal diseases including adnexitis(oophoritis, parametritis, salpingitis), fallopian tube diseases such asfallopian tube neoplasms and salpingitis, ovarian diseases (anovulation,oophoritis, ovarian cysts, polycystic ovary syndrome, premature ovarianfailure, ovarian hyperstimulation syndrome, ovarian neoplasms, Meigs'Syndrome), Parovarian cyst, endometriosis, female genital neoplasmsovarian neoplasms, uterine neoplasms, cervis neoplasms, endometrialneoplasms, vaginal neoplams, vulvar neoplasms, gynatresia, hematocolpos,hematometra, herpes genitalis, female infertility, menstruationdisorders including amenorrhea, dysmenorrhea, menorrhagia,oligomenorrhea, and premenstrual syndrome, pseudopregnancy, sexdisorders such as dypareunia and frigidity, urogenital tuberculosis,female genital tuberculosis, urogenital diseases including bladderexstrophy, epispadias, polycystic kidney (autosomal dominant andautosomal recessive), hereditary nephritis, sex differentiationdisorders including gonad dysgenesis (46 XY, Mixed), Turners' Syndrome,hermaphroditism, pseudohermaphroditism, Kallmann Syndrome, WAGRSyndrome, urogenital neoplasms, urologic neoplasms (bladder, ureteral,urethral), uterine diseases including cervix diseases (cervicitis,cervix erosion, cervix hypertrophy, cervix incompetence, cervixneoplasms), endometrial hyperplasia, endometritis, uterine hemmorrhage,menorrhagia, metrorrhagia, uterine neoplasms including cervix neoplamsand endometrial neoplasms, uterine prolapse, uterine rupture, uterineperforation, vaginal diseases including vulvovaginal candidiasis,dysparenunia, hematocolpos, leukorrhea, vaginal fistula, rectovaginalfistula, vesicovaginal fistula, vaginal neoplasms, vaginitis(trichomonas vaginitis, bacterial vaginosis, vulvovaginitis), pregnancycomplications including habitual abortion, cervix incompetence,incomplete abortion, missed abortion, septic abortion, threatenedabortion, veterinary abortion, fetal death, embryo resorption, fetalresorption, fetal diseases (chorioamnionitis, fetal erythroblastosis,hydrops fetalis, fetal alcohol syndrome, fetal anoxia, fetal distress,fetal growth retardation, fetal macrosomia, and meconium aspiration,herpes gestationis, labor complications including abruptio placentae,dystocia, uterine inertia, premature rupture of fetal membranes,chorioamnionitis, placenta accreta, placenta praevia, postpartumhemorrhage, uterine rupture, premature labor, oligohydramnios, maternalphenylketonuria, placenta diseases (abruptio placentae,chorioamnionitis, placenta accreta, placenta retained, placentalinsufficiency), polyhydramnios, cardiovascular pregnancy complications,amniotic fluid embolism, hematologic pregnancy complications, infectiouspregnancy complications (septic abortion, parasitic pregnancycomplications, puerperal infection), neoplastic pregnancy complications(trophoblastic neoplasms, choriocarcinoma, hydatidiform mole, invasivehydatidiform mole, placental site trophoblastic tumor), ectopicpregnancy, abdominal pregnancy, tubal pregnancy, pregnancy in diabetes,gestational diabetes, fetal macrosomia, pregnancy outcome, pregnancytoxemias (eclampsia, HELLP Syndrome, pre-eclamsia, EPH Gestsis,hyperemesis gravidarum), puerperal disorders, lactation disorders suchas Chiari-Frommel Syndrome, galactorrhea, and mastitis, postpartumhemorrhage, and puerperal infection.

Infertility

As stated above, KGF-2 polynucleotides, polypeptides, variants,antibodies, agonists and/or antagonists can be used to treat male orfemale infertility. Thus, in one embodiment of the invention, a methodis provided using KGF-2 polynucleotides, polypeptides, variants,antibodies, agonists and/or antagonists to treat and/or prevent maleinfertility. In another embodiment, a method is provided using KGF-2polynucleotides, polypeptides, variants, antibodies, agonists and/orantagonists to treat and/or prevent female infertility. Preferred KGF-2polypeptides used for treating infertility include KGF-2 Δ33, fulllength and mature KGF-2, KGF-2 Δ28, and polypeptides comprising aminoacids 77 to 208, 80 to 208, and 93 to 208 of KGF-2; as well as any KGF-2mutant described herein. Also preferred are polynucleotide encodingthese polypeptides.

For treatment or prevention of infertility, preferred modes ofadministration of KGF-2 include orally, rectally, parenterally,intracisternally, intradermally, intravaginally, intraperitoneally,topically (as by powders, ointments, gels, creams, drops or transdermalpatch), bucally, or as an oral or nasal spray. Other modes ofadministration are described herein. Preferably, the KGF-2polynucleotide, polypeptide, variant, antibody, agonist and/orantagonist is administered with a pharmaceutical carrier as part of apharmaceutical composition. Suitable carriers are described herein.

KGF-2 polynucleotides, polypeptides, variants, antibodies, agonistsand/or antagonists can be used to treat infertility caused by anyfactor, including environmental causes, such as coffee, MSG, plastics,Nutrasweet, alcohol, food additives, chemicals, cigarettes, pesticides,vehicle exhaust, and pollution; age; congenital infertility; low spermcount; infectious diseases, such as mumps, tuberculosis, influenza,small pox, cytomegalovirus (CMV) infection, chlamydia, mycoplasma,gonorrhea, syphilis and other sexually transmitted diseases; endocrinediseases, such as diabetes; neurological diseases, such as paraplegia;high fevers; endometriosis; toxins, such as lead in paints, varnishesand auto manufacturing agents, ethylene oxide, substances found inchemical and material industries such as paper manufacturing;chemotherapy; low weight or excessive weight loss; obesity or extremeweight gain; stress; ovulatory disorders; hormonal imbalances, CushingsSyndrome; fallopian tube blockage; pelvic infection; surgical adhesions;intrauterine devices (IUD); cervical disorders, such as anatomicalproblems, cervical infections, and mucus quality; cervical stenosis;uterine disorders, such as intrauterine adhesions, trauma to and/orinfection of the uterine lining, Asherman's Syndrome, uterine fibroids;ovarian scar tissue; ovarian cysts, including chocolate cyst;asthenospermia; maturation arrest; hypospermia; Sertoli Cell-syndrome;gonadotropin deficiency, including that arising from expanded pituitarytumors that compromises LH and FSH secretion, from surgical damage, orfrom external trauma to the cranium with damage to the portal bloodsupply; anabolic steroids; nicotine; illicit drugs, such as marijuana,heroine, and cocaine; alkaline agents, procarbozine, some halogenatedhydrocarbons used in pesticides, and frequent exposure to large amountof ethanol; pelvic inflammatory disease (PID); epididymitis; exposure totoxic substances or hazards, such as lead, cadmium, mercury, ethyleneoxide, vinyl chloride, radioactivity, and x-rays; prescription drugs forulcers or psoriasis; DES exposure in utero; exposure of the malegenitals to elevated temperatures—hot baths, whirlpools, steam rooms;hernia repair; undescended testicles; vitamin deficiency; priorabortions; and cyclophosphamide.

KGF-2 polynucleotides, polypeptides, variants, antibodies, agonistsand/or antagonists can be used to treat or prevent primary or secondaryinfertility. KGF-2 can also be used to treat temporary or permanentinfertility.

KGF-2 polynucleotides, polypeptides, variants, antibodies, agonistsand/or antagonists can be administered along with other fertilitypromoting substances, such as clomiphenne citrate (clomid, serophene),progesterone, and/or 17β-estradiol.

KGF-2 can be used to treat infertility in females during naturalconception or during assisted reproduction. Assisted reproductiontechniques include in vitro fertilization (IVF), embryo transfer (ET),gamete intrafallopian transfer (GIFT), zygote intrafallopian transfer(ZIFT), IVF with donor eggs, donor sperm, and donor embryos, andmicromanipulation of eggs and embryos. In IVF-ET, an oocyte issurgically removed, fertilized in vitro, and placed in the uterus orFallopian tube of the same woman. In oocyte donation, the oocyte isrecovered from a donor and after IVF it is transferred to an infertilerecipient as in ET. This procedure requires synchronization between thedonor and the recipient, which is generally achieved by administeringsteroid hormones to the recipient. In regular IVF-ET, the treatmentsgiven to induce multiple follicle growth often lead to insufficientluteal function. Therefore, implantation may not take place withoutsupplemental treatment with molecules such as KGF-2.

One preferred method of delivery of KGF-2 for treating or preventinginfertility in a female is through a sustained-release system via avaginal ring, as disclosed in U.S. Pat. No. 5,869,081, the disclosure ofwhich is herein incorporated by reference.

Polysiloxane carriers have been used for delivery of progesterone as acontraceptive for lactating women (Croxatto et al., 1991, in “FemaleContraception and Male Fertility Regulation. Advances in Gynecologicaland Obstetric Research Series”, Reinnebaum et al., eds.) and fordelivery of estradiol in postmenopausal women (Stumpf et al. (1982), J.Clin. Endocrinol. Metab., 58:208). Simon et al. (1986), Fertility andSterility, 46:619 disclose 17β-estradiol and/or progesterone-impregnatedpolysiloxane vaginal rings and cylinders for endometrial priming infunctionally agonadal women. The ring and cylinder system was used toachieve serum levels of 17β-estradiol and progesterone within the normalrange for an entire menstrual cycle. U.S. Pat. No. 4,816,257 disclosesthe use of polysiloxane rings containing 17β-estradiol or 17β-estradioland progesterone to mimic normal steroid hormone levels in afunctionally agonadal human female.

The present invention provides a method of administering KGF-2 for theestablishment and maintenance of pregnancy. The method of the inventioncomprises inserting a carrier containing KGF-2 into the vagina of thefemale and maintaining the carrier intravaginally for about 1–28 days.In a preferred embodiment, the carrier is a polysiloxane ring having anin vitro release rate from about 1 μg/day to 1000 mg/day, although thisamount is subject to therapeutic discretion.

Further, the method may be used to treat or prevent infertility in afemale undergoing assisted reproduction. The method comprises insertinga carrier containing KGF-2 into the vagina of a female and maintainingthe carrier intravaginally until about the seventh to twelfth week ofpregnancy. In a preferred embodiment, the carrier is a polysiloxane ringhaving an in vitro release rate of from about 1 μg/day to 1000 mg/dayKGF-2.

The present invention relates to methods for administering KGF-2 towomen with functioning ovaries and to functionally agonadal women. Womenwith functioning ovaries who are infertile or cannot conceive becausetheir partner is infertile can become pregnant through assistedreproduction techniques. However, the hormonal treatments used to inducemultiple follicle growth cause insufficient production of progesteroneby the corpus luteum. Thus, initiation and maintanence of implantationis impaired. Functionally agonadal women are infertile as a result ofundeveloped or improperly developed ovaries, surgical removal ofovaries, or other ovarian failure or dysfunction. Assisted reproductiontechniques such as OD, IVF and ET allow functionally agonadal women tobecome pregnant. However, hormone supplementation is necessary inassisted reproduction techniques in order to prepare the endometrium forthe establishment and continuation of pregnancy.

Thus, in accordance with the present invention, KGF-2 may be used totreat or prevent infertility through, inter alia, promotion of embryoimplantation. The present invention provides a method of administeringKGF-2 for the establishment and maintenance of pregnancy by assistedreproduction techniques in a normogonadal and in a functionally agonadalhuman female. The method comprises inserting a KGF-2-containing carrierinto the vagina of a normogonadal or a functionally agonadal humanfemale and maintaining the carrier intravaginally for at least abouttwenty-eight days.

The present invention also provides a method of hormone replacementtherapy for a human female undergoing assisted reproduction. The methodcomprises inserting a KGF-2-containing carrier into the vagina of ahuman female undergoing assisted reproduction and maintaining thecarrier intravaginally until about the seventh to twelfth week ofpregnancy.

The physiologically acceptable KGF-2-containing carriers useful in themethod of the present invention are preferably ring-shaped solidcarriers made of silicone rubber, also referred to herein aspolysiloxane, or other suitable material. Delivery of steroid hormonesby polysiloxane vagina rings is known in the art. The rate of passage ofKGF-2 from a polysiloxane ring is dependent upon factors including thesurface area of the ring. Accordingly, the amount of KGF-2 in the ringis conveniently described in terms of the in vitro release rate of KGF-2from the ring. In vitro release rates are routinely used in the art tocharacterize hormone-containing polysiloxane rings. KGF-2-containingpolysiloxane rings having in vitro release rates of from about 0.001 toabout 1000 mg of KGF-2 per day are contemplated for use in the presentmethod. In a preferred embodiment the polysiloxane rings have an invitro release rate of from about 0.01 to about 100 mg of KGF-2 per day.In a most preferred embodiment the polysiloxane rings have an in vitrorelease rate of about 0.1 to about 10 mg of KGF-2 per day.

The KGF-2-containing polysiloxane carriers are administered by insertioninto the vagina. The rings are inserted into the vagina and positionedaround the cervix. The ring can be inserted and removed by the femalesubject in a manner similar to that of the commonly used diaphragm, thusproviding yet another advantage of the present invention.

The KGF-2-containing carrier may be administered about two to sevendays, and preferably three days, before embryo transfer, and may besupplemented by other hormone administration, for example oraladministration of estradiol-17β or progesterone. In a preferredembodiment the carrier is a ring and is inserted three days beforeembryo transfer. The carrier is removed and replaced by another carrierafter about twenty-eight days. If pregnancy occurs, the carrier allowssufficient KGF-2 for the maintenance of pregnancy until theluteal-placental shift, at which time administration may bediscontinued. In a preferred embodiment, the ring is maintainedcontinuously in the vagina, and administration is discontinued at aboutthe twelfth week of pregnancy.

Injuries, Occupational Diseases

KGF-2 has been shown to stimulate the proliferation of a variety oftissues. Therefore, KGF-2 polynucleotides, polypeptides, agonists and/orantagonists can be used to treat injuries or occupational diseases.

Examples of injuries, occupational diseases and poisoning which can betreated or detected include occupational diseases such as agriculturalworker's diseases which include farmer's lung and silo filler's disease,bird fancier's lung, occupational dermatitis, high pressure nervoussyndrome, inert gas narcosis, laboratory infection, pneumoconiosis suchas asbestosis, berylliosis, byssinosis, Caplan's Syndrome, siderosis,silicosis such as anthracosilicosis and silicotuberculosis, poisoningsuch as alcoholic intoxication which include alcoholism such asalcoholic cardiomyopathy, fetal alcohol syndrome, alcoholic fatty liver,alcoholic hepatitis, alcoholic liver cirrhosis, alcoholic psychoses suchas alcoholic amnestic disorder, alcoholic withdrawal delirium, argyria,bites and stings such as arachnidism, insect bites and stings, snakebites, tick toxicoses such as tick paralysis, cadmium poisoning, carbontetrachloride poisoning, drug toxicity such as drug-induced akathisia,drug eruptions such as toxic epidermal necrolysis, erythema nodosum andserum sickness, drug-induced dyskinesia and neuroleptic malignantsyndrome, ergotism, fluoride poisoning, food poisoning such as botulism,favism, mushroom poisoning, salmonella food poisoning and staphylococcalfood poisoning, gas poisoning such as carbon monoxide poisoning, inertgas narcosis, toxic hepatitis, lead poisoning, mercury poisoning,mycotoxicosis such as ergotism and mushroom poisoning, overdose, plantpoisoning such as ergotism, favism, lathyrism, and milk sickness,substance-induced psychoses, wounds and injuries such as abdominalinjuries which includes traumatic diaphragmatic hernia, splenic rupturesuch as splenosis, stomach rupture, traumatic amputation, arm injuriessuch as forearm injuries which includes radius fractures and ulnafractures, humeral fractures, shoulder dislocation, shoulder fractures,tennis elbow and wrist injuries, asphyxia, athletic injuries, barotraumasuch as blast injuries and decompression sickness, birth injuries suchas obstetric paralysis, bites and stings such as human bites, burns suchas chemical burns, electric burns, inhalation burns such as smokeinhalation injury, eye burns and sunburn, contusions, dislocations suchas hip and shoulder dislocations, drowning such as near drowning,electric burns and lightning injuries, esophageal perforation,extravasation of diagnostic and therapeutic materials, foreign bodiessuch as bezoars, eye foreign bodies, foreign-body migration,foreign-body reaction such as foreign-body granuloma, fractures such asfemoral fractures such as hip fractures which includes femoral neckfractures, closed fractures, comminuted fractures, malunited fractures,open fractures, spontaneous fractures, stress fractures, ununitedfractures such as pseudarthrosis, humeral fractures, radius fracturessuch as Colles' Fractures, rib fractures, shoulder fractures, skullfractures such as jaw fractures such as mandibular and maxillaryfractures, orbital fractures and zygomatic fractures, spinal fractures,tibial fractures, ulna fractures such as Monteggia's Fractures,frostbite such as chilblains, hand injuries such as finger injuries,head injuries such as brain injuries which include brain concussion,cerebrospinal otorrhea, cerebrospinal rhinorrhea, closed head injuries,maxillofacial injuries such as facial injuries which include eyeinjuries such as eye burns, eye foreign bodies and penetrating eyeinjuries, jaw fractures such as mandibular and maxillary fractures,mandibular injuries such as mandibular fractures, and zygomaticfractures, maxillary fractures, pneumocephalus, skull fractures such asjaw fractures which includes mandibular and maxillary fractures, orbitalfractures and zygomatic fractures, heat exhaustion such as sunstroke,leg injuries such as ankle injuries, femoral fractures such as hipfractures which include femoral neck fractures, foot injuries, hipdislocation, knee injuries and tibial fractures, motion sickness such asspace motion sickness, multiple trauma, radiation injuries such asradiation-induced abnormalities, radiation-induced leukemia,radiation-induced neoplasms, osteoradionecrosis, experimental radiationinjuries, radiation pneumonitis and radiodermatitis,retropneumoperitoneum, rupture such as aortic rupture, splenic rupturesuch as splenosis, stomach rupture and uterine rupture such as uterineperforation, self mutilation, traumatic shock such as crush syndrome,soft tissue injuries, spinal cord injuries such as spinal cordcompression, spinal injuries such as spinal fractures and whiplashinjuries, sprains and strains such as repetition strain injury, tendoninjuries, thoracic injuries such as flail chest, heart injuries and ribfractures, tooth injuries such as tooth fractures which include crackedtooth syndrome, tooth luxation, tympanic membrane perforation, woundinfection, nonpenetrating wounds such as brain concussion and closedhead injuries and penetrating wounds such as penetrating eye injuries,gunshot wounds and stab wounds such as needlestick injuries.

Hemic and Lymphatic Diseases

KGF-2 polynucleotides, polypeptides, agonists, and/or antagonists can beused to treat and/or detect hemic and/or lymphatic diseases.

Examples of Hemic and Lymphatic Diseases which can be treated ordetected include aplastic anemia (such as Fanconia's Anemia), hemolyticanemia (such as autoimmune hemolytic anemia and congenital hemolyticanemia including congenital dyserythropoietic anemia, congenitalnonspherocytic hemolytic anemia, sickle cell anemia, such as hemoglobinSC disease and sickle cell trait; hereditary elliptocytosis andglucosephosphate dehydrogenase deficiency, such as favism, hemoglobin Cdisease, hereditary spherocytosis, thalassemia, such asalpha-thalassemia including hydrops fetalis, and beta-thalassemia,favism, hemoglobinuria, such as paroxysmal heboglobinuria, andhemolytic-uremic syndrome), hypochromic anemia (such as iron-deficiencyanemia), macrocytic anemia (such as megaloblastic anemia, includingpernicious anemia), myelophthisic anemia, neonatal anemia (such asfetofatal transfusion and fetomaternal transfusion), refractory anemia(such as refractory anemia with excess of blasts), sideroblastic anemia,pure red-cell aplasia, fetal erythroblastosis (such as hydrops fetalisand kernicterus), Rh Isimmunization, abetalipoproteinemia,agammaglobulinemia, dysgammaglobulinemia (such as IgA Deficiency and IgGDeficiency), hypergammaglobulinemia (such as benign monoclonalgammopathies), hyperproteinemia, paraproteinemias (such as amyloidosis,including amyloid neuropathies and cerebral amyloid angiopathy,cryoglobulinemia, heavy chain disease, such as immunoproliferative smallintestinal disease, multiple myeloma, POEMS Syndrome, Waldenstrom'sMacroglobulinemia), Protein S Deficiency.

Further examples of hemic and lymphatic diseases which can be treated ordetected include bone marrow diseases such as aplastic anemia,myelodysplastic syndromes (including refractory anemia such asrefractory anemia with excess of blasts, sideroblastic anemia,paroxysmal hemoglobinuria, and myeloid leukemia), myeloproliferativedisorders (including myelophthisic anemia, acute erythroblasticleukemia, leukemoid reaction, myelofibrosis, myeloid metaplasia,polycythemia vera, hemorrhagic thrombocythemia, and thrombocytosis),intravascular erythrocyte aggregation, hemoglobinopathies such as sicklecell anemia (including hemoglobin SC Disease and Sickle Cell Trait),Hemoglobin SC Disease, Thalassemia (including alpha-thalassemia such ashydrops fetalis, and beta thalassemia), hemorrhagic diathesis such asabrinogenemia, Christmas Disease, disseminated intravascularcoagulation, Factor VII Deficiency, Factor XI Deficiency, Factor XIIDeficiency, Factor XIII Deficiency, hemophilia, hypoprothrombinemias(including Factor V Deficiency and Factor X Deficiency), SchwartzmanPhenomenon, Bernard-Soulier Syndrome, hemolytic-urernic syndrome,platelet storage pool deficiency, thrombasthenia, hemorrhagicthrombocytopenia (including thrombocytopenic purpura such as idiopathicthrombocytopenic purpura, thrombotic thrombocytopenic purpura, andWiskott-Aldrich Syndrome), hyperglobulinemic purpura, Schoenlich-HenochPurpura, thrombocytopenic purpura (idiopathic thrombocytopenic purpura),thrombotic thrombocytopenic purpura, Wiskott-Aldrich Syndrome,hereditary hemorrhagic telangiectasia, vitamin K Deficiency (includinghemorrhagic disease of newborn), and von Willebrand's Disease, leukocytedisorders such as eosinophilia (including angiolymphoid hyperplasia witheosinophilia, eosinophilia-myalgia syndrome, eosinophilic granuloma, andhypereosinophilic syndrome such as pulmonary eosinophilia), infectiousmononucleosis, leukocytosis (including leukamoid reaction andlymphocytosis), leukopenia (including agranulocytosis such asneutropenia, and lymphopenia such as idiopathic CD4-PositiveT-Lymphopenia), Pelger-Huet Anomaly, phagocyte bactericidal dysfunction(including Chediak-Higashi Syndrome, Chronic Granulomatous Disease,Job's Syndrome), methemoglobinemia, pancytopenia, polycythemia,hematologic, preleukemia, and sulfhemoglobinemia.

Additional examples of hemic and lymphatic diseases which can be treatedor detected include lymphatic diseases such as lymphadenitis (includingcat-scratch disease and mesenteric lymphadenitis), lymphangiectasis,lymphangitis, lymphedema (including elephantiasis and filarialelephantiasis), lymphocele, lymphoproliferative disorders (includingagammaglobulinemia, amyloidosis such as amyloid neuropathies andcerebral amyloid angiopathy, giant lymph node hyperplasia, heavy chaindisease such as immunoproliferative small intestinal disease,immunoblastic lymphadenopathy, infectious mononucleosis, hairy cellleukemia, lymphocytic leukemia, myeloid leukemia (including acutenonlymphocytic leukemia and acute myelocytic leukemia), lymphangiomyoma(including lymphangiomyomatosis), and lymphoma (including Hodgkin'sDisease, Non-Hodgkin's Lymphoma such as B-Cell Lymphoma includingBurkitt's Lymphoma, AIDS-Related Lymphoma, mucosa-associated lymphoidtissue lymphoma, and small-cell lymphoma, diffuse lymphoma includingdiffuse large-cell lymphoma, immunoblastic large-cell lymphoma,lymphoblastic lymphoma, diffuse mixed-cell lymphoma, small lymphocyticlymphoma, and small noncleaved-cell lymphoma, follicular lymphomaincluding follicular large-cell lymphoma, follicular mixed-celllymphoma, and follicular small cleaved-cell lymphoma, high-gradelymphoma including immunoblastic large-cell lymphoma, lymphoblasticlymphoma, and small noncleaved-cell lymphoma such as Burkitt's Lymphoma,intermediate-grade lymphoma including diffuse large-cell lymphoma,follicular large-cell lymphoma, diffuse mixed-cell lymphoma, and diffusesmall cleaved-cell lymphoma, large-cell lymphoma including diffuselarge-cell lymphoma, follicular large-cell lymphoma, immunoblasticlarge-cell lymphoma, Ki-1 large-cell lymphoma, and immunoblasticlarge-cell lymphoma, low-grade lymphoma including follicular mixed-celllymphoma, mucosa-associated lymphoid tissue, follicular smallcleaved-cell lymphoma, and small lymphocytic lymphoma, mixed-celllymphoma including diffuse mixed-cell lymphoma and follicular mixed-celllymphoma, small-cell lymphoma including diffuse small-cleaved celllymphoma, follicular small cleaved-cell lymphoma, small lymphocyticlymphoma, and small noncleaved-cell lymphoma, t-cell lymphoma includinglymphoblastic lymphoma, cutaneous T-cell lymphoma such as Ki-1large-cell lymphoma, fungoides mycosis, and Sezary Syndrome, andperipheral T-cell lymphoma, undifferentiated lymphoma including diffuselarge-cell lymphoma, and small noncleaved-cell lymphoma such asBurkitt's Lymphoma, lymphomatoid granulomatosis), Marek's Disease,sarcoidosis (including pulmonary sarcoidosis and uveoparotid Fever),tumor lysis syndrome, mucocutaneous lymph node syndrome,reticuloendotheliosis (including Gaucher's Disease, histiocytosis suchas malignant histiocytic disorders including malignant histiocytosis,acute monocytic leukemia, large-cell lymphoma such as Ki-1 Large-CellLymphoma, Langerhans-Cell Histiocytosis such as Eosinophilic Granuloma,Hand-Scheller-Christian Syndrome, and Letterer-Siwe Disease,Non-Langerhans-Cell Histiocytosis such as Sinus Histiocytosis,Niemann-Pick Disease, Sea-Blue Histiocyte Syndrome, and JuvenileXanthogranuloma, Mast-Cell Sarcoma), Splenic Diseases (includingHypersplenism, Myeloid Metaplasia, Splenic Infarction, SplenicNeoplasms, Splenic Rupture such as Splenosis, Splenomegaly, and SplenicTuberculosis), Thymus Hyperplasia, Thymus Neoplasms, Lymph NodeTuberculosis such as King's Evil.

Neonatal Diseases and Abnormalities

KGF-2 polynucleotides, polypeptides, agonists and/or antagonists can beused to treat, prevent, and/or detect neonatal diseases and/orabnormalities.

Examples of neonatal diseases and abnormalities which can be treated ordetected include drug-induced abnormalities, multiple abnormalitiesincluding Alagille Syndrome, Angelman Syndrome, basal cell nevussyndrome, Beckwith-Widemann Syndrome, Bloom Syndrome, Bonnevie-UlrichSyndrome, Cockayne Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome,Down Syndrome, Ectodermal Dysplasia such as Ellis-Van Creveld Syndromeand Focal Dermal Hypoplasia, Gardner Syndrome, holoprosencephaly,incontinentia pigmenti, Laurence-Moon Biedl Syndrome, Marfan Syndrome,Nail-Patella Syndrome, Oculocerebrorenal Syndrome, OrofaciodigitalSyndromes, Prader-Willi Syndrome, Proteus Syndrome, Prune BellySyndrome, Congenital Rubella Syndrome, Rubenstein-Taybi Syndrome, ShortRib-Polydactyly Syndrome, Waardenburg's Syndrome, Wolfram Syndrome,Zelweger Syndrome, Radiation-Induced Abnormalities, Chromosomeabnormalities including Angelman Syndrome, Beckwith-Wiedemann,Cri-du-Chat Syndrome, Down Syndrome, holoprosencephaly, Prader-WilliSyndrome, sex chromosome abnormalities such as Bonnevie-Ulrich Syndrome,Ectodermal Dysplasia including Focal Dermal Hypoplasia, Fragile XSyndrome, 46,XY Gonadal Dysgenesis, Mixed Gonadal Dysgenesis, KallmanSyndrome, Klinefelter's Syndrome, Oculocerebrorenal Syndrome,Orofaciodigital Syndromes, Turner's Syndrome, and XYY Karyotype, anddigestive system abnormalities.

Respiratory Diseases

KGF-2 has been shown to stimulate proliferation of cells of therespiratory tract. Thus, KGF-2 polynucleotides, polypeptides, agonistsand/or antagonists can be used to treat and/or detect respiratorydiseases.

Examples of respiratory tract diseases which can be treated or detectedinclude bronchial diseases, such as asthma (including exercise-inducedasthma and status asthmaticus) bronchial fistula, bronchialhyperreactivity, bronchial neoplasms, bronchial spasm, bronchiectasis,bronchitis (including bronchiolitis, bronchiolitis obliterans,organizing pneumonia, viral bronchiolitis, bronchogenic cyst,bronchopneumonia, tracheobronchomegaly), ciliary motility disorders suchas Kartagener's Syndrome, laryngeal diseases (such as laryngealgranuloma, laryngeal edema, laryngeal neoplasms, laryngealperichondritis, laryngismus, laryngitis such as croup, laryngostenosis,laryngeal tuberculosis, vocal cord paralysis, voice disorders such asaphonia and hoarseness), lung diseases, such as atelectasis whichincludes middle lobe syndrome, bronchopulomonary dysplasia, congenitalcystic adenomatoid malformation of lung, cystic fibrosis, pulmonaryplasma cell granuloma, hemoptysis, lung abscess, fungal lung diseasessuch as allergic bronchopulmonary aspergillosis and Pneumocystis cariniipneumonia, interstitial lung diseases such as extrinsic allergicalveolitis such as Bird Fancier's Lung, Farmer's Lung, Goodpasture'sSyndrome, langerhans-cell histiocytosis, pneumoconiosis such asasbestosis, berylliosis, byssinosis, Caplan's Syndrome, siderosis,silicosis such as anthracosilicosis and silicotuberculosis, pulmonaryfibrosis, radiation pneumonitis, pulmonary sarcoidosis, Wegener'sGranulomatosis), obstructive lung diseases, viral bronchiolitis,pulmonary emphysema, parasitic lung diseases such as pulmonaryechinococcosis, lung neoplasms such as bronchogenic carcinoma, pulmonarycoin lesion and Pancoast's Syndrome, Meconium Aspiration, Pneumonia(such as bronchopneumonia, pleuropneumonia, aspiration pneumonia such aslipid pneumonia, bacterial pneumonia such as lobar pneumonia, MycoplasmaPneumonia, Rickettsial Pneumonia and Staphylococcal Pneumonia,Pneumocystis carinii pneumonia, viral pneumonia), pulmonary alveolarproteinosis, pulmonary edema, pulmonary embolism, pulmonaryeosinophilia, pulmonary veno-occlusive disease, respiratory distresssyndrome such as hyaline membrane disease, adult respiratory distresssyndrome, Scimitar Syndrome, Silo Filler's Disease, Pulmonarytuberculosis such as silicotuberculosis; nose diseases, such as choanalatresia, epistaxis, lethal midline granuloma, nasal obstruction, nasalpolyps, acquired nose deformities, nose neoplasms such as nasal polyps,paranasal sinus neoplasms such as maxillary sinus neoplasms, paranasalsinus neoplasms such as maxillary sinus neoplasms, sinusitis such asethmoid sinusitis, frontal sinusitis, maxillary sinusitis and sphenoidsinusitis, rhinitis such as hay fever, perennial allergic rhinitis,atrophic rhinitis and vasomotor rhinitis, rhinoscleroma).

Respiratory disease which may be treated and/or diagnosed also includepleural diseases, such as chylothorax, pleural empyema (such astuberculous empyema), hemopneumothorax, hemothorax, hydropneumothorax,hydrothorax, pleural effusion such as malignant pleural effusion,pleural neoplasms such as malignant pleural effusion, pleurisy such aspleuropneumonia, pneumothorax, pleural tuberculosis such as tuberculousempyema, respiration disorders such as apnea such as sleep apneasyndromes which include Pickwickian Syndrome, Cheyne-Stokes Respiration,cough, dyspnea such as paroxysmal dyspnea, hoarseness, hyperventilationsuch as respiratory alkalosis, laryngismus, meconium aspiration, mouthbreathing, respiratory distress syndrome such as hyaline membranedisease, adult respiratory distress syndrome, respiratory insufficiencysuch as respiratory acidosis, airway obstruction such as nasalobstruction, laryngeal granuloma, hantavirus pulmonary syndrome,hypoventilation, intrinsic positive-pressure respiration and respiratoryparalysis, respiratory hypersensitivity such as extrinsic allergicalveolitis such as Bird Fancier's Lung and Farmer's Lung, allergicbronchopulomary aspergillosis, asthma such as exercise-induced asthmaand status asthmaticus, hay fever, perennial allergic rhinitis,respiratory system abnormalities such as bronchogenic cyst,bronchopulmonary sequestration, choanal atresia, congenital cysticadenomatoid malformation of lung, Kartagener's Syndrome, ScimitarSyndrome, tracheobronchomegaly, respiratory tract fistula such asbronchial fistula which includes tracheoesophageal fistula), respiratorytract infections (such as bronchitis which includes bronchiolitis suchas viral bronchiolitis, common cold, pleural empyema such as tuberculousempyema, influenza, laryngitis such as epiglottitis, legionellosis suchas Legionnaries' Disease, Lung Abscess, Pleurisy such asPleuropneumonia, Pneumonia such as Bronchopneumonia, Pleuropneumonia,Aspiration Pneumonia such as Lipid Pneumonia, Bacterial Pneumonia suchas Lobar Pneumonia, Mycoplasma Pneumonia, Rickettsial Pneumonia andStaphylococcal Pneumonia, Pneumocystis carinii Pneumonia, ViralPneumonia, Rhinitis, Rhinoscleroma, Sinusitis such as Ethmoid Sinusitis,Frontal Sinusitis, Maxillary Sinusitis and Sphenoid Sinusitis,Tonsillitis such as Peritonsillar Abscess, Tracheitis, LaryngealTuberculosis, Pleural Tuberculosis such as Tuberculous Empyema,Pulmonary Tuberculosis such as Silicotuberculosis, Whooping Cough,Respiratory Tract Neoplasms such as Bronchial Neoplasms, LaryngealNeoplasms, Lung Neoplasms such as Bronchogenic Carcinoma, Pulmonary CoinLesion and Pancoast's Syndrome, Nose Neoplasms such as Nasal Polyps,Paranasal Sinus Neoplasms such as Maxillary Sinus Neoplasms, PleuralNeoplasms such as Malignant Pleural Effusion, Tracheal Neoplasms,Tracheal Diseases such as Tracheal Neoplasms, Tracheal Stenosis,Tracheitis, Tracheobronchomegaly and Tracheoesophageal Fistula.

Examples of Otorhinolaryngologic Diseases which can be treated ordetected include Ciliary Motility Disorders such as Kartagener'sSyndrome, Ear Diseases such as Middle Ear Cholesteatoma, Acquired EarDeformities, Ear Neoplasms, Earache, Hearing Disorders such as Deafnesswhich include Sudden Deafness, Partial Hearing Loss such as BilateralHearing Loss, Conductive Hearing Loss, Functional Hearing Loss,High-Frequency Hearing Loss, Sensorineural Hearing Loss such as CentralHearing Loss, Noise-Induced Hearing Loss and Presbycusis, LoudnessRecruitment, Tinnitus, Herpes Zoster Oticus, Labyrinth Diseases such asCochlear Diseases, Endolymphatic Hydrops such as Meniere's Disease,Labyrinthitis, Vestibular Diseases such as Motion Sickness whichincludes Space Motion Sickness, Vertigo, Otitis such as Otitis Externa,Otitis Media such as Mastoiditis, Otitis Media with Effusion andSuppurative Ottitis Media, Otosclerosis, Retrocochlear Diseases such asAcoustic Nerve Diseases which include Acoustic Neuroma such asNeurofibromatosis 2, Central Auditory Diseases such as AuditoryPerceptual Disorders and Central Hearing Loss, Tympanic MembranePerforation), Laryngeal Diseases such as Laryngeal Granuloma, LaryngealEdema, Laryngeal Neoplasms, Laryngeal Perichondritis, Laryngismus,Laryngitis such as Croup, Laryngostenosis, Laryngeal Tuberculosis, VocalCord Paralysis, Voice Disorders such as Aphonia and Hoarseness, NoseDiseases (such as Choanal Atresia, Epistaxis, Lethal Midline Granuloma,Nasal Obstruction, Nasal Polyps, Acquired Nose Deformities, NoseNeoplasms such as Nasal Polyps, Paranasal Sinus Neoplasms such asMaxillary Sinus Neoplasms, Paranasal Sinus Diseases such as ParanasalSinus Neoplams which include Maxillary Sinus Neoplasms, Sinusitis suchas Ethmoid Sinusitis, Frontal Sinusitis, Maxillary Sinusitis andSphenoid Sinusitis, Rhinitis such as Hay Fever, Perennial AllergicRhinitis, Atrophic Rhinitis and Vasomotor Rhinitis, Rhinoscleroma),otorhinolaryngologic neoplasms such as ear neoplasms, laryngealneoplasms, acoustic neuroma such as Neurofibromatosis 2, nose neoplasmssuch as nasal polyps, paranasal sinus neoplasms such as maxillary sinusneoplasms, pharyngeal neoplasms such as hypopharyngeal neoplasms,nasopharyngeal neoplasms, oropharyngeal neoplasms such as tonsillarneoplasms, pharyngeal neoplasms such as hypopharyngeal neoplasms,nasopharyngeal neoplasms, oropharyngeal neoplasms which includestonsillar neoplasms, pharyngitis, retropharyngeal abscess, tonsillitis,and velopharyngeal insufficiency.

Neurologic Diseases

KGF-2 polynucleotides, polypeptides, agonists and/or antagonists may beused to treat and/or detect neurologic diseases.

Examples of neurologic diseases which can be treated or detected includebrain diseases (such as metabolic brain diseases which includesphenylketonuria such as maternal phenylketonuria, pyruvate carboxylasedeficiency, pyruvate dehydrogenase complex deficiency, Wernicke'sEncephalopathy, brain edema, brain neoplasms such as cerebellarneoplasms which include infratentorial neoplasms, cerebral ventricleneoplasms such as choroid plexus neoplasms, hypothalamic neoplasms,supratentorial neoplasms, canavan disease, cerebellar diseases such ascerebellar ataxia which include spinocerebellar degeneration such asataxia telangiectasia, cerebellar dyssynergia, Friederich's Ataxia,Machado-Joseph Disease, olivopontocerebellar atrophy, cerebellarneoplasms such as infratentorial neoplasms, diffuse cerebral sclerosissuch as encephalitis periaxialis, globoid cell leukodystrophy,metachromatic leukodystrophy and subacute sclerosing panencephalitis,cerebrovascular disorders (such as carotid artery diseases which includecarotid artery thrombosis, carotid stenosis and Moyamoya Disease,cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,cerebral arteriosclerosis, cerebral arteriovenous malformations,cerebral artery diseases, cerebral embolism and thrombosis such ascarotid artery thrombosis, sinus thrombosis and Wallenberg's Syndrome,cerebral hemorrhage such as epidural hematoma, subdural hematoma andsubarachnoid hemorrhage, cerebral infarction, cerebral ischemia such astransient cerebral ischemia, Subclavian Steal Syndrome andvertebrobasilar insufficiency, vascular dementia such as multi-infarctdementia, periventricular leukomalacia, vascular headache such ascluster headache, migraine, dementia such as AIDS Dementia Complex,presenile dementia such as Alzheimer's Disease and Creutzfeldt-JakobSyndrome, senile dementia such as Alzheimer's Disease and progressivesupranuclear palsy, vascular dementia such as multi-infarct dementia,encephalitis which include encephalitis periaxialis, viral encephalitissuch as epidemic encephalitis, Japanese Encephalitis, St. LouisEncephalitis, tick-borne encephalitis and West Nile Fever, acutedisseminated encephalomyelitis, meningoencephalitis such asuveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease andsubacute sclerosing panencephalitis, encephalomalacia such asperiventricular leukomalacia, epilepsy such as generalized epilepsywhich includes infantile spasms, absence epilepsy, myoclonic epilepsywhich includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsysuch as complex partial epilepsy, frontal lobe epilepsy and temporallobe epilepsy, post-traumatic epilepsy, status epilepticus such asEpilepsia Partialis Continua, Hallervorden-Spatz Syndrome, hydrocephalussuch as Dandy-Walker Syndrome and normal pressure hydrocephalus,hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria,narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebripseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebraltoxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, centralnervous system infections such as AIDS Dementia Complex, Brain Abscess,subdural empyema, encephalomyelitis such as Equine Encephalomyelitis,Venezuelan Equine Encephalomyelitis, Necrotizing HemorrhagicEncephalomyelitis, Visna, cerebral malaria, meningitis such asarachnoiditis, aseptic meningtitis such as viral meningtitis whichincludes lymphocytic choriomeningitis. Bacterial meningtitis whichincludes Haemophilus Meningtitis, Listeria Meningtitis, MeningococcalMeningtitis such as Waterhouse-Friderichsen Syndrome, PneumococcalMeningtitis and meningeal tuberculosis, fungal meningitis such asCryptococcal Meningtitis, subdural effusion, meningoencephalitis such asuvemeningoencephalitic syndrome, myelitis such as transverse myelitis,neurosyphilis such as tabes dorsalis, poliomyelitis which includesbulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases(such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy,Gerstmann-Straussler Syndrome, Kuru, Scrapie) cerebral toxoplasmosis,central nervous system neoplasms such as brain neoplasms that includecerebellear neoplasms such as infratentorial neoplasms, cerebralventricle neoplasms such as choroid plexus neoplasms, hypothalamicneoplasms and supratentorial neoplasms, meningeal neoplasms, spinal cordneoplasms which include epidural neoplasms, demyelinating diseases suchas Canavan Diseases, diffuse cerebral sceloris which includesadrenoleukodystrophy, encephalitis periaxialis, globoid cellleukodystrophy, diffuse cerebral sclerosis such as metachromaticleukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagicencephalomyelitis, progressive multifocal leukoencephalopathy, multiplesclerosis, central pontine myelinolysis, transverse myelitis,neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue Syndrome,Visna, High Pressure Nervous Syndrome, Meningism, spinal cord diseasessuch as amyotonia congenita, amyotrophic lateral sclerosis, spinalmuscular atrophy such as Werdnig-Hoffmann Disease, spinal cordcompression, spinal cord neoplasms such as epidural neoplasms,syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mental retardationsuch as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome,Down Syndrome, Gangliosidoses such as gangliosidoses G(M1), SandhoffDisease, Tay-Sachs Disease, Hartnup Disease, homocystinuria,Laurence-Moon-Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup UrineDisease, mucolipidosis such as fucosidosis, neuronalceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria suchas maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome,Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervoussystem abnormalities such as holoprosencephaly, neural tube defects suchas anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity,encephalocele, meningocele, meningomyelocele, spinal dysraphism such asspina bifida cystic a and spin a bifida occulta, hereditary motor andsensory neuropathies which include Charcot-Marie Disease, Hereditaryoptic atrophy, Refsum's Disease, hereditary spastic paraplegia,Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathiessuch as Congenital Analgesia and Familial Dysautonomia, Neurologicmanifestations (such as agnosia that include Gerstmann's Syndrome,Amnesia such as retrograde amnesia, apraxia, neurogenic bladder,cataplexy, communicative disorders such as hearing disorders thatincludes deafness, partial hearing loss, loudness recruitment andtinnitus, language disorders such as aphasia which include agraphia,anomia, broca aphasia, and Wernicke Aphasia, Dyslexia such as AcquiredDyslexia, language development disorders, speech disorders such asaphasia which includes anomia, broca aphasia and Wernicke Aphasia,articulation disorders, communicative disorders such as speech disorderswhich include dysarthria, echolalia, mutism and stuttering, voicedisorders such as aphonia and hoarseness, decerebrate state, delirium,fasciculation, hallucinations, meningism, movement disorders such asangelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia,muscle hypotonia, myoclonus, tic, torticollis and tremor, musclehypertonia such as muscle rigidity such as stiff-man syndrome, musclespasticity, paralysis such as facial paralysis which includes HerpesZoster Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such asdiplopia, Duane's Syndrome, Homer's Syndrome, Chronic progressiveexternal ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis,Tropical Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome,quadriplegia, respiratory paralysis and vocal cord paralysis, paresis,phantom limb, taste disorders such as ageusia and dysgeusia, visiondisorders such as amblyopia, blindness, color vision defects, diplopia,hemianopsia, scotoma and subnormal vision, sleep disorders such ashypersomnia which includes Kleine-Levin Syndrome, insomnia, andsomnambulism, spasm such as trismus, unconsciousness such as coma,persistent vegetative state and syncope and vertigo, neuromusculardiseases such as amyotonia congenita, amyotrophic lateral sclerosis,Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscularatrophy such as spinal muscular atrophy, Charcot-Marie Disease andWerdnig-Hoffmann Disease, Postpoliomyelitis Syndrome, MuscularDystrophy, Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita,Nemaline Myopathy, Familial Periodic Paralysis, MultiplexParamyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome,peripheral nervous system diseases such as acrodynia, amyloidneuropathies, autonomic nervous system diseases such as Adie's Syndrome,Barre-Lieou Syndrome, Familial Dysautonomia, Homer's Syndrome, ReflexSympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseasessuch as Acoustic Nerve Diseases such as Acoustic Neuroma which includesNeurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders which includesamblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia suchas Duane's Syndrome, Horner's Syndrome, Chronic Progressive ExternalOphthalmoplegia which includes Kearns Syndrome, Strabismus such asEsotropia and Exotropia, Oculomotor Nerve Paralysis, Optic NerveDiseases such as Optic Atrophy which includes Hereditary Optic Atrophy,Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica,Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, DemyelinatingDiseases such as Neuromyelitis Optica and Swayback, Diabeticneuropathies such as diabetic foot, nerve compression syndromes such ascarpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndromesuch as cervical rib syndrome, ulnar nerve compression syndrome,neuralgia such as causalgia, cervico-brachial neuralgia, facialneuralgia and trigeminal neuralgia, neuritis such as experimentalallergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritisand radiculities such as polyradiculitis, hereditary motor and sensoryneuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy,Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-HoffmannDisease, Hereditary Sensory and Autonomic Neuropathies which includeCongenital Analgesia and Familial Dysautonomia, POEMS Syndrome,Sciatica, Gustatory Sweating and Tetany).

Metabolic and Endocrine Diseases

KGF-2 polynucleotides, polypeptides, agonists and/or antagonists may beused to treat and/or diagnose metabolic or endocrine diseases.

Examples of nutritional and metabolic diseases which can be treated ordetected include achlorhydria, acid-base imbalance, acidosis (includinglactic, renal tubular, or respiratory), diabetic ketoacidosis, ketosis,alkalosis, respiratory alkalosis, calcium metabolism sisorders,calcinosis, calciphylaxis, CREST syndrome, nephrocalcinosis, pathologicdecalcification, hypercalcemia, hypocalcemia, tetany, osteomalacia,pseudohypoparathyroidism, Rickets, diabetes insipidus, nephrogenicdiabetes insipidus, Wolfram Syndrome, diabetes mellitus (includingexperimental and insulin-dependent, lipoatrophic,non-insulin-dependent), diabetic angiopathies, diabetic foot,gestational diabetes, fetal macrosomia, glucose intolerance, glycosuria,renal glycosuria, hyperglycemia, hyperlipidemia, hypercholesterolemia,hyperlipoproteinemia, hypertriglyceridemia, hyperprolactinemia,hypervitaminosis A, hypoglycemia, insulin coma, malabsorption syndromes(including Blind Loop Syndrome, Celiac Disease, lactose intolerance,intestinal lipodystrophy, Tropical Sprue), inborn errors in metabolism(including inborn errors in amino acid metabolism, ocular albinism,oculocutaneous albinism, piebaldism), alkaptonuria, ochronosis, renalaminoaciduria, cystinuria, Hartnup Disease, homocystinuria, Maple SyrupUrine Disease, multiple carboxylase deficiency, phenylketonuria,maternal phenylketonuria, amyloidosis, amyloid neuropathies, cerebralamyloid angiopathy, inborn errors in carbohydrate metabolism such asinborn errors in fructose metabolism (Fructose-1,6-DiphosphataseDeficiency, fuctose intolerance), galactosemia, glucose intolerance,glycogen storage disease (Types I, II, III, IV, V, VI, VII, VIII),hyperoxaluria, primary hyperoxalura, mannosidosis, mucopolysaccharidoses(I, II, III, IV, VI, VII), multiple carboxylase deficiency, inbornerrors in pyruvate metabolism, Leigh Disease, pyruvate carboxylasedeficiency, pyruvate dehydrogenase complex deficiency, glucosephosphatedehadrogenase deficiency, hereditary hyperbilirubinemia, Crigler-NajjarSyndrome, Gilbert's Disease, chronic idiopathic jaundice, inborn errorsin lipid metabolism such as hyperlipoproteinemia, familialhypercholestrolemia, familial combined hyperlipidemia,hypercholesterolemia (familial, Type III, IV, V), familial lipoproteinlipase deficiency, hypolipoproteinemia (abetalipoproteinemia,hypobetalipoproteinemia, lecithin acyltransferase deficiency, TangierDisease), lipoidosis (cholesterol ester storage disease,lipoidproteinosis, neuronal ceroid-lipofuscinosis, Refsum's Disease,Sjogren-Larsson Syndrome, sphingolipidoses (adrenoleukodystrophy,Fabry's Disease, ganglisidoses, Sandhoff Disease, Tay-Sachs Disease,Gaucher's Disease, globoid cell leukodystrophy, metachromaticleukodystrophy, Niemann-Pick Disease, Sea-Blue Histiocyte Syndrome,Wolman Disease, mitochrondrial myopathies, mitochorondrialencephalomyopathies, MELAS Syndrome, MERRF Syndrome, external chronicprogressive ophthalmoplegia, lysosomal storage diseases such ascholestrol ester storage disease, mannosidosis, mucolipidosis,fucosidosis, muchopolysaccharidosis (I, II, III, IV, VI, and VII),inborn errors in metal metabolism including hemochromatosis,hepatolenticular degeneration, hypophosphatasia, familialhypophosphatemia, kinky hair syndrome, familial periodic paralysis, andpseudohypoparathyroidism, mucolipidosis, fucosidosis, porphyria,(erythroheatic, erythropoietic, hepatic, acute intermittent, cutaneatarda), inborn errors in purine-pyrimidine metabolism such as gout,gouty arthritis, and lesch-Nyhan Syndrome, inborn errors in renaltubular transport such as renal tubular acidosis, renal aminoaciduria,cystinuria, hartnup disease, cystinosis, Fanconi Syndrome, renalgylycosuria, familial hypophosphatemia, oculocerbrorenal syndrome, andpseudohypoaldosteronism, phosphorus metabolism disorders,hypophosphatemia, protein-losing enteropathies, intestinallymphangiectasis, water-electrolyte imbalance (dehydration,hypercalcemia, hyperkalemia, hypernatremia, hypocalcemia, hyponatremia,inappropriate adh syndrome, water intoxication), xanthomatosis, WolmanDisease, Child nutrition disorders such as infant nutrition disorders,deficiency diseases such as avitaminosis, ascorbic acid deficiency,scurvy, vitamin A deficiency, vitamin B deficiency, choline deficiency,folic acid deficiency, pellagra, pyridoxine deficiency, riboflavindeficiency, thiamine deficiency, beriberi, Wernicke's Encephalopathy,vitamin B₁₂ deficiency (anemia, pernicious), vitamin D deficiency,(osteomalacia, steatitis), vitamin E deficiency (steatitis), vitamin Kdeficiency, magnesium deficiency, potassium deficiency, proteindeficiency (protein-energy malnutrition, kwashiorkor), swayback, obesityin diabetes, morbid obesity, Pickwickian Syndrome, Prader-WilliSyndrome, and starvation.

Examples of endocrine diseases which can be treated or detected includeadrenal gland diseases (cortex diseases, nortex neoplasms), adrenalgland hyperfunction (Cushing's Syndrome, hyperaldosteronism, Bartter'sDisease), adrenal gland hypofunction (Addison's Disease,adrenoleukodystrophy, hypoaldosteronism), adrenal gland neoplasms,adrenal cortex neoplasms, congenital adrenal hyperplasia,Waterhouse-Friderichsen Syndrome, breast neoplasms, male breastneoplasms, fibrocystic disease of the breast, gynecomastia, lactationdisorders such as Chiari-Frommel Syndrome and galactorrhea, mastitis,Bowie mastitis, diabetes mellitus (experimental, insulin-dependent,Wolfram Syndrome, lipoatrophic, and non-insulin dependent), diabeticangiopathies, diabetic foot, diabetic retinopathy, diabetic coma,hyperglycemic hyperosmolar nonketotic coma, diabetic ketoacidosis,diabetic nephropathies and that associated with diabetic foot, obesityin diabetes, gestational diabetes, fetal macrosomia, dwarfism (CockayneSyndrome, pituitary, thanatophoric dysplasia), endocrine gland neoplasmssuch as adrenal cortex neoplasma, multiple endocrine neoplasia (types 1,2a, 2b), neoplastic endocrine-like syndromes, ACTH syndrome (ectopic),Zollinger-Ellison Syndrome, Ovarian neoplasms, Meig's Syndrome,parathyroid neoplasms, pituitary neoplasms, Nelson Syndrome, TesticularNeoplasms, thymus neoplasms, thyroid neoplasms, thyroid nodule, gonadaldisorders such as adrenal hyperplasia (congenital), feminization,testicular feminization, hyperandrogenism, hypogonadism, eunuchism,Kallmann Syndrome, Kinefelter's Syndrome, ovarian diseases such asanovulation, oophoritis, ovarian cysts, polycystic ovary syndrome,premature ovarian failure, ovarian hyperstimulation syndrome, ovarianneoplasms, Meigs' Syndrome, delayed puberty, and precocious puberty, sexdifferentiation disorders such as gonadal dysgenesis (46,XY, mixed) andTurner's Syndrome, hermaphroditism, pseudohermaphroditism, KallmannSyndrome, Klinefelter's Syndrome, Testicular feminization, testiculardiseases such as Cryptorchidism, Orchitis, testicular neoplasms,virilism, hirsutism, hyperinsulinism, neoplastic endocrine-likesyndromes such as ACTH Syndrome (Ectopic) and Zollinger-EllisonSyndrome, parathyroid diseases including hyperparathyroidism(secondary), renal osteodystrophy, hypoparathyroidism, tetany,parathyroid neoplasms, pituitary diseases, Empy Sella Syndrome,hyperpituitarism, acromegaly, gigantism, hypopituitarism (diabetesinsipidus, nephrogenic disbetes insipidus, Wolfram Syndrome, pituitarydwarfism), inappropriate ADH syndrome, pituitary apoplexy, pituitaryneoplasms, Nelson Syndrome, autoimmune polyendocrinopathies, progeria,Werner's Syndrome, thymus hyperplasia, thyroid diseases such aseuthyroid sick syndromes, goiter (endemic, nodular, substernal, Graves'Disease), hyperthyroidism and that associated with Graves' Disease,hyperthyroxinemia, hypothyroidism (cretinism and myxedema), thyroidhormone resistance syndrome, thyroid neoplasms, thyroid nodule,thyroiditis (autoimmune, subacute, suppurative), thyrotoxicosis, thyroidcrisis, and endocrine tuberculosis.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition ofapoptosis that could be treated or detected by KGF-2 polynucleotides orpolypeptides, as well as antagonists or agonists of KGF-2, includecancers (such as follicular lymphomas, carcinomas with p53 mutations,and hormone-dependent tumors, including, but not limited to coloncancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer);autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome,Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, KGF-2 polynucleotides, polypeptides, and/orantagonists of the invention are used to inhibit growth, progression,and/or metasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by KGF-2 polynucleotides orpolypeptides, or agonists or antagonists of KGF-2, include, but are notlimited to, progression, and/or metastases of malignancies and relateddisorders such as leukemia (including acute leukemias (e.g., acutelymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis that could be treated ordetected by KGF-2 polynucleotides or polypeptides, as well as agonistsor antagonists of KGF-2, include AIDS; neurodegenerative disorders (suchas Alzheimer's disease, Parkinson's disease, Amyotrophic lateralsclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumoror prior associated disease); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) myelodysplastic syndromes (such as aplastic anemia), graft v.host disease, ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), liver injury (e.g.,hepatitis related liver injury, ischemia/reperfusion injury, cholestosis(bile duct injury) and liver cancer); toxin-induced liver disease (suchas that caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing KGF-2 polynucleotides orpolypeptides, as well as agonists or antagonists of KGF-2, fortherapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.KGF-2 polynucleotides or polypeptides, as well as agonists orantagonists of KGF-2, may be clinically useful in stimulating woundhealing including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resultingfrom heat exposure or chemicals, and other abnormal wound healingconditions such as uremia, malnutrition, vitamin deficiencies andcomplications associted with systemic treatment with steroids, radiationtherapy and antineoplastic drugs and antimetabolites. KGF-2polynucleotides or polypeptides, as well as agonists or antagonists ofKGF-2, could be used to promote dermal reestablishment subsequent todermal loss.

KGF-2 polynucleotides or polypeptides, as well as agonists orantagonists of KGF-2, could be used to increase the adherence of skingrafts to a wound bed and to stimulate re-epithelialization from thewound bed. The following are types of grafts that KGF-2 polynucleotidesor polypeptides, agonists or antagonists of KGF-2, could be used toincrease adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepidermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft,) heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, and thick split graft. KGF-2polynucleotides or polypeptides, as well as agonists or antagonists ofKGF-2, can also be used to promote skin strength and to improve theappearance of aged skin.

It is believed that KGF-2 polynucleotides or polypeptides, as well asagonists or antagonists of KGF-2, will also produce changes inhepatocyte proliferation, and epithelial cell proliferation in the lung,breast, pancreas, stomach, small intestine, and large intestine. KGF-2polynucleotides or polypeptides, as well as agonists or antagonists ofKGF-2, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. KGF-2 polynucleotides or polypeptides, agonists or antagonists ofKGF-2, may promote proliferation of endothelial cells, keratinocytes,and basal keratinocytes.

KGF-2 polynucleotides or polypeptides, as well as agonists orantagonists of KGF-2, could also be used to reduce the side effects ofgut toxicity that result from radiation, chemotherapy treatments orviral infections. KGF-2 polynucleotides or polypeptides, as well asagonists or antagonists of KGF-2, may have a cytoprotective effect onthe small intestine mucosa. KGF-2 polynucleotides or polypeptides, aswell as agonists or antagonists of KGF-2, may also stimulate healing ofmucositis (mouth ulcers) that result from chemotherapy and viralinfections.

KGF-2 polynucleotides or polypeptides, as well as agonists orantagonists of KGF-2, could further be used in full regeneration of skinin full and partial thickness skin defects, including burns, (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands),treatment of other skin defects such as psoriasis. KGF-2 polynucleotidesor polypeptides, as well as agonists or antagonists of KGF-2, could beused to treat epidermolysis bullosa, a defect in adherence of theepidermis to the underlying dermis which results in frequent, open andpainful blisters by accelerating reepithelialization of these lesions.KGF-2 polynucleotides or polypeptides, as well as agonists orantagonists of KGF-2, could also be used to treat gastric and doudenalulcers and help heal by scar formation of the mucosal lining andregeneration of glandular mucosa and duodenal mucosal lining morerapidly. Inflammatory bowel diseases, such as Crohn's disease andulcerative colitis, are diseases which result in destruction of themucosal surface of the small or large intestine, respectively. Thus,KGF-2 polynucleotides or polypeptides, as well as agonists orantagonists of KGF-2, could be used to promote the resurfacing of themucosal surface to aid more rapid healing and to prevent progression ofinflammatory bowel disease. Treatment with KGF-2 polynucleotides orpolypeptides, agonists or antagonists of KGF-2, is expected to have asignificant effect on the production of mucus throughout thegastrointestinal tract and could be used to protect the intestinalmucosa from injurious substances that are ingested or following surgery.KGF-2 polynucleotides or polypeptides, as well as agonists orantagonists of KGF-2, could be used to treat diseases associated withthe under expression of KGF-2.

Moreover, KGF-2 polynucleotides or polypeptides, as well as agonists orantagonists of KGF-2, could be used to prevent and heal damage to thelungs due to various pathological states. A growth factor such as KGF-2polynucleotides or polypeptides, as well as agonists or antagonists ofKGF-2, which could stimulate proliferation and differentiation andpromote the repair of alveoli and brochiolar epithelium to prevent ortreat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treatedusing KGF-2 polynucleotides or polypeptides, agonists or antagonists ofKGF-2. Also, KGF-2 polynucleotides or polypeptides, as well as agonistsor antagonists of KGF-2, could be used to stimulate the proliferation ofand differentiation of type II pneumocytes, which may help treat orprevent disease such as hyaline membrane diseases, such as infantrespiratory distress syndrome and bronchopulmonary displasia, inpremature infants.

KGF-2 polynucleotides or polypeptides, as well as agonists orantagonists of KGF-2, could stimulate the proliferation anddifferentiation of hepatocytes and, thus, could be used to alleviate ortreat liver diseases and pathologies such as fulminant liver failurecaused by cirrhosis, liver damage caused by viral hepatitis and toxicsubstances (i.e., acetaminophen, carbon tetrachloride and otherhepatotoxins known in the art).

In addition, KGF-2 polynucleotides or polypeptides, as well as agonistsor antagonists of KGF-2, could be used treat or prevent the onset ofdiabetes mellitus. In patients with newly diagnosed Types I and IIdiabetes, where some islet cell function remains, KGF-2 polynucleotidesor polypeptides, as well as agonists or antagonists of KGF-2, could beused to maintain the islet function so as to alleviate, delay or preventpermanent manifestation of the disease. Also, KGF-2 polynucleotides orpolypeptides, as well as agonists or antagonists of KGF-2, could be usedas an auxiliary in islet cell transplantation to improve or promoteislet cell function.

Infectious Disease

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, can be used to treat or detect infectious agents. For example, byincreasing the immune response, particularly increasing theproliferation and differentiation of B and/or T cells, infectiousdiseases may be treated. The immune response may be increased by eitherenhancing an existing immune response, or by initiating a new immuneresponse. Alternatively, KGF-2 polynucleotides or polypeptides, oragonists or antagonists of KGF-2, may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated or detected by KGF-2 polynucleotides orpolypeptides, or agonists or antagonists of KGF-2. Examples of viruses,include, but are not limited to the following DNA and RNA viralfamilies: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus,Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae,Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as,Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g.,Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxyiridae(such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus),Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g.,Rubivirus). Viruses falling within these families can cause a variety ofdiseases or symptoms, including, but not limited to: arthritis,bronchiollitis, encephalitis, eye infections (e.g., conjunctivitis,keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, ChronicActive, Delta), meningitis, opportunistic infections (e.g., AIDS),pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles,Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. KGF-2 polynucleotides or polypeptides, or agonists orantagonists of KGF-2, can be used to treat or detect any of thesesymptoms or diseases.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated or detected by KGF-2 polynucleotides orpolypeptides, or agonists or antagonists of KGF-2, include, but notlimited to, the following Gram-Negative and Gram-positive bacterialfamilies and fungi: Actinomycetales (e.g., Corynebacterium,Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax,Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia,Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis,Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella,Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter,Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae(e.g., Acinetobacter, Gonorrhea, Menigococcal), PasteurellaceaInfections (e.g., Actinobacillus, Heamophilus, Pasteurella),Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, andStaphylococcal. These bacterial or fungal families can cause thefollowing diseases or symptoms, including, but not limited to:bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis,uveitis), gingivitis, opportunistic infections (e.g., AIDS relatedinfections), paronychia, prosthesis-related infections, Reiter'sDisease, respiratory tract infections, such as Whooping Cough orEmpyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery,Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea,meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,and wound infections. KGF-2 polynucleotides or polypeptides, or agonistsor antagonists of KGF-2, can be used to treat or detect any of thesesymptoms or diseases.

Moreover, parasitic agents causing disease or symptoms that can betreated or detected by KGF-2 polynucleotides or polypeptides, oragonists or antagonists of KGF-2, include, but not limited to, thefollowing families: Amebiasis, Babesiosis, Coccidiosis,Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis,Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas. These parasites can cause a variety ofdiseases or symptoms, including, but not limited to: Scabies,Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,giardiasis), liver disease, lung disease, opportunistic infections(e.g., AIDS related), Malaria, pregnancy complications, andtoxoplasmosis. KGF-2 polynucleotides or polypeptides, or agonists orantagonists of KGF-2, can be used to treat or detect any of thesesymptoms or diseases.

Preferably, treatment using KGF-2 polynucleotides or polypeptides, oragonists or antagonists of KGF-2, could either be by administering aneffective amount of KGF-2 polypeptide to the patient, or by removingcells from the patient, supplying the cells with KGF-2 polynucleotide,and returning the engineered cells to the patient (ex vivo therapy).Moreover, the KGF-2 polypeptide or polynucleotide can be used as anantigen in a vaccine to raise an immune response against infectiousdisease.

Regeneration

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, can be used to differentiate, proliferate, and attract cells,leading to the regeneration of tissues. (See, Science 276:59–87 (1997).)The regeneration of tissues could be used to repair, replace, or protecttissue damaged by congenital defects, trauma (wounds, burns, incisions,or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, KGF-2 polynucleotides or polypeptides, or agonists orantagonists of KGF-2, may increase regeneration of tissues difficult toheal. For example, increased tendon/ligament regeneration would quickenrecovery time after damage. KGF-2 polynucleotides or polypeptides, oragonists or antagonists of KGF-2, of the present invention could also beused prophylactically in an effort to avoid damage. Specific diseasesthat could be treated include of tendinitis, carpal tunnel syndrome, andother tendon or ligament defects. A further example of tissueregeneration of non-healing wounds includes pressure ulcers, ulcersassociated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by usingKGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, to proliferate and differentiate nerve cells. Diseases that couldbe treated using this method include central and peripheral nervoussystem diseases, neuropathies, or mechanical and traumatic disorders(e.g., spinal cord disorders, head trauma, cerebrovascular disease, andstoke). Specifically, diseases associated with peripheral nerveinjuries, peripheral neuropathy (e.g., resulting from chemotherapy orother medical therapies), localized neuropathies, and central nervoussystem diseases (e.g., Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, and Shy-Dragersyndrome), could all be treated using the KGF-2 polynucleotides orpolypeptides, or agonists or antagonists of KGF-2.

Chemotaxis

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, may have chemotaxis activity. A chemotaxic molecule attracts ormobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells,mast cells, eosinophils, epithelial and/or endothelial cells) to aparticular site in the body, such as inflammation, infection, or site ofhyperproliferation. The mobilized cells can then fight off and/or healthe particular trauma or abnormality.

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, may increase chemotaxic activity of particular cells. Thesechemotactic molecules can then be used to treat inflammation, infection,hyperproliferative disorders, or any immune system disorder byincreasing the number of cells targeted to a particular location in thebody. For example, chemotaxic molecules can be used to treat wounds andother trauma to tissues by attracting immune cells to the injuredlocation. As a chemotactic molecule, KGF-2 could also attractfibroblasts, which can be used to treat wounds.

It is also contemplated that KGF-2 polynucleotides or polypeptides, oragonists or antagonists of KGF-2, may inhibit chemotactic activity.These molecules could also be used to treat disorders. Thus, KGF-2polynucleotides or polypeptides, or agonists or antagonists of KGF-2,could be used as an inhibitor of chemotaxis.

Binding Activity

KGF-2 polypeptides may be used to screen for molecules that bind toKGF-2 or for molecules to which KGF-2 binds. The binding of KGF-2 andthe molecule may activate (agonist), increase, inhibit (antagonist), ordecrease activity of the KGF-2 or the molecule bound. Examples of suchmolecules include antibodies, oligonucleotides, proteins (e.g.,receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand ofKGF-2, e.g., a fragment of the ligand, or a natural substrate, a ligand,a structural or functional mimetic. (See, Coligan et al., CurrentProtocols in Immunology 1(2):Chapter 5 (1991).) Similarly, the moleculecan be closely related to the natural receptor to which KGF-2 binds, orat least, a fragment of the receptor capable of being bound by KGF-2(e.g., active site). In either case, the molecule can be rationallydesigned using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express KGF-2, either as a secreted protein oron the cell membrane. Preferred cells include cells from mammals, yeast,Drosophila, or E. coli. Cells expressing KGF-2(or cell membranecontaining the expressed polypeptide) are then preferably contacted witha test compound potentially containing the molecule to observe binding,stimulation, or inhibition of activity of either KGF-2 or the molecule.

The assay may simply test binding of a candidate compound to KGF-2,wherein binding is detected by a label, or in an assay involvingcompetition with a labeled competitor. Further, the assay may testwhether the candidate compound results in a signal generated by bindingto KGF-2.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining KGF-2, measuring KGF-2/molecule activity or binding, andcomparing the KGF-2/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure KGF-2 level or activity in asample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure KGF-2 level or activity by eitherbinding, directly or indirectly, to KGF-2 or by competing with KGF-2 fora substrate.

Additionally, the receptor to which KGF-2 binds can be identified bynumerous methods known to those of skill in the art, for example, ligandpanning and FACS sorting (Coligan, et al., Current Protocols in Immun.,1(2), Chapter 5, (1991)). For example, expression cloning is employedwherein polyadenylated RNA is prepared from a cell responsive to thepolypeptides, for example, NIH3T3 cells which are known to containmultiple receptors for the FGF family proteins, and SC-3 cells, and acDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to thepolypeptides. Transfected cells which are grown on glass slides areexposed to the polypeptide of the present invention, after they havebeen labelled. The polypeptides can be labeled by a variety of meansincluding iodination or inclusion of a recognition site for asite-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clone that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of KGF-2 therebyeffectively generating agonists and antagonists of KGF-2. See generally,U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724–33(1997); Harayama, S. Trends Biotechnol. 16(2):76–82 (1998); Hansson, L.O., et al., J. Mol. Biol. 287:265–76 (1999); and Lorenzo, M. M. andBlasco, R. Biotechniques 24(2):308–13 (1998) (each of these patents andpublications are hereby incorporated by reference). In one embodiment,alteration of KGF-2 polynucleotides and corresponding polypeptides maybe achieved by DNA shuffling. DNA shuffling involves the assembly of twoor more DNA segments into a desired KGF-2 molecule by homologous, orsite-specific, recombination. In another embodiment, KGF-2polynucleotides and corresponding polypeptides may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of KGF-2 may be recombined with one or more components,motifs, sections, parts, domains, fragments, etc. of one or moreheterologous molecules. In preferred embodiments, the heterologousmolecules are fibroblast growth factor family members. In furtherpreferred embodiments, the heterologous molecule is a growth factor suchas, for example, platelet-derived growth factor (PDGF), insulin-likegrowth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermalgrowth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bonemorphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins Aand B, decapentaplegic (dpp), 60A, OP-2, dorsalin, growthdifferentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1,TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor(GDNF).

Other preferred fragments are biologically active KGF-2 fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the KGF-2 polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, the polypeptide of the present invention, thecompound to be screened and ³[H] thymidine under cell culture conditionswhere the fibroblast cell would normally proliferate. A control assaymay be performed in the absence of the compound to be screened andcompared to the amount of fibroblast proliferation in the presence ofthe compound to determine if the compound stimulates proliferation bydetermining the uptake of ³[H] thymidine in each case. The amount offibroblast cell proliferation is measured by liquid scintillationchromatography which measures the incorporation of ³[H] thymidine. Bothagonist and antagonist compounds may be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the KGF-2 receptor is measured and the ability of thecompound to bind to the receptor and elicit a second messenger responseis measured to determine if the compound is a potential agonist orantagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting the KGF-2/molecule.Moreover, the assays can discover agents which may inhibit or enhancethe production of KGF-2 from suitably manipulated cells or tissues.

Therefore, the invention includes a method of identifying compoundswhich bind to KGF-2 comprising the steps of: (a) incubating a candidatebinding compound with KGF-2; and (b) determining if binding hasoccurred. Moreover, the invention includes a method of identifyingagonists/antagonists comprising the steps of: (a) incubating a candidatecompound with KGF-2, (b) assaying a biological activity, and (c)determining if a biological activity of KGF-2 has been altered.

Also, one could identify molecules bind KGF-2 experimentally by usingthe beta-pleated sheet regions disclosed in FIG. 4 and Table 1.Accordingly, specific embodiments of the invention are directed topolynucleotides encoding polypeptides which comprise, or alternativelyconsist of, the amino acid sequence of each beta pleated sheet regionsdisclosed in FIG. 4/Table 1. Additional embodiments of the invention aredirected to polynucleotides encoding KGF-2 polypeptides which comprise,or alternatively consist of, any combination or all of the beta pleatedsheet regions disclosed in FIG. 4/Table 1. Additional preferredembodiments of the invention are directed to polypeptides whichcomprise, or alternatively consist of, the KGF-2 amino acid sequence ofeach of the beta pleated sheet regions disclosed in FIG. 4/Table 1.Additional embodiments of the invention are directed to KGF-2polypeptides which comprise, or alternatively consist of, anycombination or all of the beta pleated sheet regions disclosed in FIG.4/Table 1.

Antisense And Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:1, or the complementary strand thereof, and/or to nucleotidesequences contained in the deposited clone 75977. In one embodiment,antisense sequence is generated internally by the organism, in anotherembodiment, the antisense sequence is separately administered (see, forexample, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotidesas Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed for example, in Okano, J., Neurochem.56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the KGF-2 antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the KGF-2 antisense nucleic acid. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others know inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding KGF-2, or fragments thereof, can beby any promoter known in the art to act in vertebrate, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude, but are not limited to, the SV40 early promoter region(Bernoist and Chambon, Nature 29:304–310 (1981), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.,Cell 22:787–797 (1980), the herpes thymidine promoter (Wagneret al.,Proc. Natl. Acad. Sci. U.S.A. 78:1441–1445 (1981), the regulatorysequences of the metallothionein gene (Brinster, et al., Nature296:39–42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a KGF-2gene. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded KGF-2 antisense nucleic acids, a single strandof the duplex DNA may thus be tested, or triplex formation may beassayed. The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid Generally,the larger the hybridizing nucleic acid, the more base mismatches with aKGF-2 RNA it may contain and still form a stable duplex (or triplex asthe case may be). One skilled in the art can ascertain a tolerabledegree of mismatch by use of standard procedures to determine themelting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333–335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of KGF-2 shown in FIGS. 1A-B couldbe used in an antisense approach to inhibit translation of endogenousKGF-2 mRNA. Oligonucleotides complementary to the 5′ untranslated regionof the mRNA should include the complement of the AUG start codon.Antisense oligonucleotides complementary to mRNA coding regions are lessefficient inhibitors of translation but could be used in accordance withthe invention. Whether designed to hybridize to the 5′-, 3′- or codingregion of KGF-2 mRNA, antisense nucleic acids should be at least sixnucleotides in length, and are preferably oligonucleotides ranging from6 to about 50 nucleotides in length. In specific aspects, theoligonucleotide is at least 10 nucleotides, at least 17 nucleotides, atleast 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553–6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648–652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958–976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539–549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625–6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131–6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327–330).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448–7451),. etc.

While antisense nucleotides complementary to the KGF-2 coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222–1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy KGF-2 mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585–591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of KGF-2 (FIGS. 1A–B). Preferably, the ribozymeis engineered so that the cleavage recognition site is located near the5′ end of the KGF-2 mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express KGF-2 invivo. DNA constructs encoding the ribozyme may be introduced into thecell in the same manner as described above for the introduction ofantisense encoding DNA. A preferred method of delivery involves using aDNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous KGF-2 messages and inhibit translation.Since ribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat the diseasesdescribed herein.

Other Activities

The polypeptide of the present invention, as a result of the ability tostimulate vascular endothelial cell growth, may be employed in treatmentfor stimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. These polypeptide may also be employed tostimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed tostimulate neuronal growth and to treat and prevent neuronal damage whichoccurs in certain neuronal disorders or neuro-degenerative conditionssuch as Alzheimer's disease, Parkinson's disease, and AIDS-relatedcomplex. KGF-2 may have the ability to stimulate chondrocyte growth,therefore, they may be employed to enhance bone and periodontalregeneration and aid in tissue transplants or bone grafts.

The polypeptide of the present invention may be also be employed toprevent skin aging due to sunburn by stimulating keratinocyte growth.

The KGF-2 polypeptide may also be employed for preventing hair loss,since FGF family members activate hair-forming cells and promotesmelanocyte growth. Along the same lines, the polypeptides of the presentinvention may be employed to stimulate growth and differentiation ofhematopoietic cells and bone marrow cells when used in combination withother cytokines.

The KGF-2 polypeptide may also be employed to maintain organs beforetransplantation or for supporting cell culture of primary tissues.

The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, may also be used to modulate mammalian characteristics, such asbody height, weight, hair color, eye color, skin, percentage of adiposetissue, pigmentation, size, and shape (e.g., cosmetic surgery).Similarly, KGF-2 polynucleotides or polypeptides, or agonists orantagonists of KGF-2, may be used to modulate mammalian metabolismaffecting catabolism, anabolism, processing, utilization, and storage ofenergy.

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, may be used to change a mammal's mental state or physical stateby influencing biorhythms, caricadic rhythms, depression (includingdepressive disorders), tendency for violence, tolerance for pain,reproductive capabilities (preferably by Activin or Inhibin-likeactivity), hormonal or endocrine levels, appetite, libido, memory,stress, or other cognitive qualities.

KGF-2 polynucleotides or polypeptides, or agonists or antagonists ofKGF-2, may also be used as a food additive or preservative, such as toincrease or decrease storage capabilities, fat content, lipid, protein,carbohydrate, vitamins, minerals, cofactors or other nutritionalcomponents.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976–985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087–3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (⁹⁹Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ⁹⁹mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLE 1 Bacterial Expression and Purification of KGF-2

The DNA sequence encoding KGF-2, ATCC® #75977, is initially amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′ endsequences of the processed KGF-2 cDNA (including the signal peptidesequence). The 5′ oligonucleotide primer has the sequence: 5′CCCCACATGTGGAAATGGATACTGACACATTGTGCC 3′ (SEQ ID No. 3) contains anAflIII restriction enzyme site including and followed by 30 nucleotidesof KGF-2 coding sequence starting from the presumed initiation codon.The 3′ sequence: 5′ CCCAAGCTTCCACAAACGTTGCCTTCCTCTATGAG 3′ (SEQ ID No.4) contains complementary sequences to HindIII site and is followed by26 nucleotides of KGF-2. The restriction enzyme sites are compatiblewith the restriction enzyme sites on the bacterial expression vectorpQE-60 (Qiagen, Inc. Chatsworth, Calif.). pQE-60 encodes antibioticresistance (Amp^(r)), a bacterial origin of replication (ori), anIPTG-regulatable promoter operator (P/0), a ribosome binding site (RBS),a 6-His tag and restriction enzyme sites. pQE-60 is then digested withNcoI and HindIII. The amplified sequences are ligated into pQE-60 andare inserted in frame. The ligation mixture is then used to transform E.coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described inSambrook, J., et al., Molecular Cloning: A Laboratory Manual, ColdSpring Laboratory Press, (1989). M15/rep4 contains multiple copies ofthe plasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kan^(r)). Transformants are identified by theirability to grow on LB plates and ampicillin/kanamycin resistant coloniesare selected. Plasmid DNA is isolated and confirmed by restrictionanalysis. Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) is then added to a finalconcentration of 1 mM. IPTG interacts with the lacI repressor to causeit to dissociate from the operator, forcing the promoter to directtranscription. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedKGF-2 is purified from this solution by chromatography on a Heparinaffinity column under conditions that allow for tight binding of theproteins (Hochuli, E., et al., J. Chromatography 411:177–184 (1984)).KGF-2 (75% pure) is eluted from the column by high salt buffer.

EXAMPLE 2 Bacterial Expression and Purification of a Truncated Versionof KGF-2

The DNA sequence encoding KGF-2, ATCC® #75977, is initially amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the truncated version of the KGF-2 polypeptide. Thetruncated version comprises the polypeptide minus the 36 amino acidsignal sequence, with a methionine and alanine residue being added justbefore the cysteine residue which comprises amino acid 37 of thefull-length protein. The 5′ oligonucleotide primer has the sequence 5′CATGCCATGGCGTGCCAAGCCCTTGGTCAGGACATG 3′ (SEQ ID NO:5) contains an NcoIrestriction enzyme site including and followed by 24 nucleotides ofKGF-2 coding sequence. The 3′ sequence 5′ CCCAAGCTTCCACAAACGTTGC CTTCCTCTATGAG 3′ (SEQ ID NO:6) contains complementary sequences to HindIII siteand is followed by 26 nucleotides of the KGF-2 gene. The restrictionenzyme sites are compatible with the restriction enzyme sites on thebacterial expression vector pQE-60 (Qiagen, Inc., Chatsworth, Calif.).pQE-60 encodes antibiotic resistance (Ampr), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/0), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-60 is then digested with NcoI and HindIII. The amplified sequencesare ligated into pQE-60 and are inserted in frame. The ligation mixtureis then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by theprocedure described in Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989). M15/rep4contains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kanr). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis. Clones containing thedesired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedKGF-2 is purified from this solution by chromatography on a Heparinaffinity column under conditions that allow for tight binding theproteins (Hochuli, E. et al., J. Chromatography 411:177–184 (1984)).KGF-2 protein is eluted from the column by high salt buffer.

EXAMPLE 3 Cloning and Expression of KGF-2 Using the BaculovirusExpression System

The DNA sequence encoding the full length KGF-2 protein, ATCC® #75977,is amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the gene:

The 5′ primer has the sequence 5′ GCGGGATCCGCCATCATGTGGAAATGGATACTCAC 3′(SEQ ID NO:7) and contains a BamHI restriction enzyme site (in bold)followed by 6 nucleotides resembling an efficient signal for theinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.,196:947–950 (1987)) and just behind the first 17 nucleotides of theKGF-2 gene (the initiation codon for translation “ATG” is underlined).

The 3′ primer has the sequence 5′ GCGCGGTACCACAAACGTTGCCTTCCT 3′ (SEQ IDNO:8) and contains the cleavage site for the restriction endonucleaseAsp718 and 19 nucleotides complementary to the 3′ non-translatedsequence of the KGF-2 gene. The amplified sequences are isolated from a1% agarose gel using a commercially available kit from Qiagen, Inc.,Chatsworth, Calif. The fragment is then digested with the endonucleasesBamHI and Asp718 and then purified again on a 1% agarose gel. Thisfragment is designated F2.

The vector pA2 (modification of pVL941 vector, discussed below) is usedfor the expression of the KGF-2 protein using the baculovirus expressionsystem (for review see: Summers, M. D. & Smith, G. E., A manual ofmethods for baculovirus vectors and insect cell culture procedures,Texas Agricultural Experimental Station Bulletin No. 1555 (1987)). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhidrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamHI andAsp718. The polyadenylation site of the simian virus (SV) 40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used such as pAc373, pVL941 and pAcIM1(Luckow, V. A. & Summers, M. D., Virology, 170:31–39).

The plasmid is digested with the restriction enzymes BamHI and Asp718.The DNA is then isolated from a 1% agarose gel using the commerciallyavailable kit (Qiagen, Inc., Chatsworth, Calif.). This vector DNA isdesignated V2.

Fragment F2 and the plasmid V2 are ligated with T4 DNA ligase. E. coliHB101 cells are then transformed and bacteria identified that containedthe plasmid (pBacKGF-2) with the KGF-2 gene using PCR with both cloningoligonucleotides. The sequence of the cloned fragment is confirmed byDNA sequencing.

5 μg of the plasmid pBacKGF-2 is co-transfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner, et al., Proc. Natl. Acad. Sci. USA, 84:7413–7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacKGF-2 aremixed in a sterile well of a microtiter plate containing 50 μl of serumfree Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture is added drop-wise to the Sf9 insect cells (ATCC® CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate is rocked back and forth to mix the newly addedsolution. The plate is then incubated for 5 hours at 27° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plate is put back into an incubator and cultivation continued at 27°C. for four days.

After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9–10).

Four days after the serial dilution, the viruses are added to the cellsand blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf 9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-KGF-2 at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μCi of ³⁵S methionine and 5 μCi ³⁵S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins are visualized bySDS-PAGE and autoradiography.

EXAMPLE 4

Most of the vectors used for the transient expression of the KGF-2protein gene sequence in mammalian cells should carry the SV40 origin ofreplication. This allows the replication of the vector to high copynumbers in cells (e.g., COS cells) which express the T antigen requiredfor the initiation of viral DNA synthesis. Any other mammalian cell linecan also be utilized for this purpose.

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV,HTLVI, HIVI and the immediate early promoter of the cytomegalovirus(CMV). However, cellular signals can also be used (e.g., human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC® 37152), pSV2dhfr (ATCC®37146) and pBC12MI (ATCC® 67109). Mammalian host cells that could beused include, human Hela, 283, H9 and Jurkart cells, mouse NIH3T3 andC127 cells, Cos 1, Cos 7 and CV1, African green monkey cells, quailQC1–3 cells, 293T cells, mouse L cells and Chinese hamster ovary cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, hygromycin allows theidentification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) is a usefulmarker to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277–279 (1991); Bebbington et al., Bio/Technology 10:169–175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) cells are often used for the production ofproteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology,438–447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521–530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

A. Expression of Recombinant KGF-2 in COS Cells

The expression of plasmid, KGF-2 HA was derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. The HAtag correspond to an epitope derived from the influenza hemagglutininprotein as previously described (Wilson, I., et al., Cell 37:767,(1984)). The infusion of HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope. A DNA fragment encoding the entire KGF-2 precursor HAtag fused in frame with the HA tag, therefore, the recombinant proteinexpression is directed under the CMV promoter.

The plasmid construction strategy is described as follows:

The DNA sequence encoding KGF-2, ATCC® #75977, is constructed by PCRusing two primers: the 5′ primer

5′ TAACGAGGATCCGCCATCATGTGGAAATGGATACTGACAC 3′ (SEQ ID NO:9) contains aBamHI site followed by 22 nucleotides of KGF-2 coding sequence startingfrom the initiation codon; the 3′ sequence

5′ TAAGCACTCGAGTGAGTGTACCACCATTGGAAGAAATG 3′ (SEQ ID NO:10) containscomplementary sequences to an XhoI site, HA tag and the last 26nucleotides of the KGF-2 coding sequence (not including the stop codon).Therefore, the PCR product contains a BamHI site, KGF-2 coding sequencefollowed by an XhoI site, an HA tag fused in frame, and a translationtermination stop codon next to the HA tag. The PCR amplified DNAfragment and the vector, pcDNA-3′ HA, are digested with BamHI and XhoIrestriction enzyme and ligated resulting in pcDNA-3′ HA-KGF-2. Theligation mixture is transformed into E. coli strain XL1 Blue (StratageneCloning Systems, La Jolla, Calif.) the transformed culture is plated onampicillin media plates and resistant colonies are selected. Plasmid DNAwas isolated from transformants and examined by PCR and restrictionanalysis for the presence of the correct fragment. For expression of therecombinant KGF-2, COS cells were transfected with the expression vectorby DEAE-DEXTRAN method (Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989)). The expressionof the KGF-2 HA protein was detected by radiolabelling andimmunoprecipitation method (Harlow, E. & Lane, D., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cellswere labelled for 8 hours with 35S-cysteine two days post transfection.Culture media were then collected and cells were lysed with detergent(RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mMTris, pH 7.5) (Wilson, I., et al., Id. 37:767 (1984)). Both cell lysateand culture media were precipitated with a HA specific monoclonalantibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.

B: Expression and purification of human KGF-2 protein using the CHOExpression System

The vector pC1 is used for the expression of KFG-2 protein. Plasmid pC1is a derivative of the plasmid pSV2-dhfr [ATCC® Accession No. 37146].Both plasmids contain the mouse DHFR gene under control of the SV40early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357–1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107–143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol.9:64–68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene it is usually co-amplified and over-expressed. It is stateof the art to develop cell lines carrying more than 1,000 copies of thegenes. Subsequently, when the methotrexate is withdrawn, cell linescontain the amplified gene integrated into the chromosome(s).

Plasmid pC1 contains for the expression of the gene of interest a strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438–4470)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521–530, 1985).Down stream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, PvuII,and NruI. Behind these cloning sites the plasmid contains translationalstop codons in all three reading frames followed by the 3′ intron andthe polyadenylation site of the rat preproinsulin gene. Other highefficient promoters can also be used for the expression, e.g., the humanβ-actin promoter, the SV40 early or late promoters or the long terminalrepeats from other retroviruses, e.g., HIV and HTLVI. For thepolyadenylation of the mRNA other signals, e.g., from the human growthhormone or globin genes can be used as well.

Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC1 is digested with the restriction enzyme BamHI and thendephosphorylated using calf intestinal phosphates by procedures known inthe art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding KFG-2, ATCC® No. 75977, is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene:

The 5′ primer has the sequence: 5′ TAACGAGGATCCGCCATCATGTGGAAATGGATACTGACAC 3′ (SEQ ID NO:9) containing the underlined BamHIrestriction enzyme site followed by 21 bases of the sequence of KGF-2 ofFIGS. 1A–1C (SEQ ID NO:1). Inserted into an expression vector, asdescribed below, the 5′ end of the amplified fragment encoding humanKGF-2 provides an efficient signal peptide. An efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947–950 (1987) is appropriately located in thevector portion of the construct.

The 3′ primer has the sequence: 5′ TAAGCAGGATCCTGAGTGTACCACCATTGGAAGAAATG 3′ (SEQ ID NO:10) containing the BamHI restrictionfollowed by nucleotides complementary to the last 26 nucleotides of theKGF-2 coding sequence set out in FIGS. 1A–1C (SEQ ID NO:1), notincluding the stop codon.

The amplified fragments are isolated from a 1% agarose gel as describedabove and then digested with the endonuclease BamHI and then purifiedagain on a 1% agarose gel.

The isolated fragment and the dephosphorylated vector are then ligatedwith T4 DNA ligase. E. coli HB101 cells are then transformed andbacteria identified that contain the plasmid pC1. The sequence andorientation of the inserted gene is confirmed by DNA sequencing.

Transfection of CHO-DHFR-Cells

Chinese hamster ovary cells lacking an active DHFR enzyme are used fortransfection. 5 μg of the expression plasmid C1 are cotransfected with0.5 μg of the plasmid pSVneo using the lipofecting method (Felgner etal., supra). The plasmid pSV2-neo contains a dominant selectable marker,the gene neo from Tn5 encoding an enzyme that confers resistance to agroup of antibiotics including G418. The cells are seeded in alpha minusMEM supplemented with 1 mg/ml G418. After 2 days, the cells aretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany)and cultivated for 10–14 days. After this period, single clones aretrypsinized and then seeded in 6-well petri dishes using differentconcentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (500 nM, 1 μM, 2 μM, 5 μM). The same procedure isrepeated until clones grow at a concentration of 100 μM.

The expression of the desired gene product is analyzed by Western blotanalysis and SDS-PAGE.

EXAMPLE 5 Transcription and Translation of Recombinant KGF-2 in vitro

A PCR product is derived from the cloned cDNA in the pA2 vector used forinsect cell expression of KGF-2. The primers used for this PCR were: 5′

ATTAACCCTCACTAAAGGGAGGCCATGTGGAAATGGATACT GACACATTGTGCC 3′ (SEQ IDNO:11) and

5′ CCCAAGCTTCCACAAACGTTGCCTTCCTCTATGAG 3′ (SEQ ID NO:12).

The first primer contains the sequence of a T3 promoter 5′ to the ATGinitiation codon. The second primer is complimentary to the 3′ end ofthe KGF-2 open reading frame, and encodes the reverse complement of astop codon.

The resulting PCR product is purified using a commercially available kitfrom Qiagen. 0.5 μg of this DNA is used as a template for an in vitrotranscription-translation reaction. The reaction is performed with a kitcommercially available from Promega under the name of TNT. The assay isperformed as described in the instructions for the kit, usingradioactively labeled methionine as a substrate, with the exception thatonly 1/2 of the indicated volumes of reagents are used and that thereaction is allowed to proceed at 33° C. for 1.5 hours.

Five μl of the reaction is electrophoretically separated on a denaturing10 to 15% polyacrylamide gel. The gel is fixed for 30 minutes in amixture of water:Methanol:Acetic acid at 6:3:1 volumes respectively. Thegel is then dried under heat and vacuum and subsequently exposed to anX-ray film for 16 hours. The film is developed showing the presence of aradioactive protein band corresponding in size to the conceptuallytranslated KGF-2, strongly suggesting that the cloned cDNA for KGF-2contains an open reading frame that codes for a protein of the expectedsize.

EXAMPLE 6 Expression Via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature overnight. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219–25 (1988)) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer containing an EcoRI site and the 3′ primerfurther includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

EXAMPLE 7 KGF-2 Stimulated Wound Healing in the Diabetic Mouse Model

To demonstrate that keratinocyte growth factor-2 (KGF-2) wouldaccelerate the healing process, the genetically diabetic mouse model ofwound healing was used. The full thickness wound healing model in thedb+/db+mouse is a well characterized, clinically relevant andreproducible model of impaired wound healing. Healing of the diabeticwound is dependent on formation of granulation tissue andre-epithelialization rather than contraction (Gartner, M. H. et al., J.Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol.136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283–293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1–7 (1983); Leiter et al., Am. J. of Pathol. 114:46–55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221–232 (1984); Robertson et al., Diabetes29(1):60–67 (1980); Giacomelli et al., Lab Invest. 40(4):460–473 (1979);Coleman, D. L., Diabetes 31 (Suppl):1–6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375–1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235–1246 (1990)). Theresults of this study demonstrated that KGF-2 has a potent stimulatoryeffect on the healing of full thickness wounds in diabetic andnon-diabetic heterozygous littermates. Marked effects onre-epithelialization and an increase in collagen fibrils, granulationtissue within the dermis were observed in KGF-2 treated animals. Theexogenous application of growth factors may accelerate granulationtissue formation by drawing inflammatory cells into the wound.

Animals

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates were used in this study(Jackson Laboratories). The animals were purchased at 6 weeks of age andwere 8 weeks old at the beginning of the study. Animals wereindividually housed and received food and water ad libitum. Allmanipulations were performed using aseptic techniques. The experimentswere conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

KGF-2

The recombinant human KGF-2 used for the wound healing studies wasover-expressed and purified from pQE60-Cys37, an E. coli expressionvector system (pQE-9, Qiagen). The protein expressed from this constructis the KGF-2 from Cysteine at position 37 to Serine at position 208 witha 6×(His) tag attached to the N-terminus of the protein (SEQ IDNOS:29–30) (FIG. 15). Fractions containing greater than 95% purerecombinant materials were used for the experiment. Keratinocyte growthfactor-2 was formulated in a vehicle containing 100 mM Tris, 8.0 and 600mM NaCl. The final concentrations were 80 μg/mL and 8 μg/mL of stocksolution. Dilutions were made from stock solution using the samevehicle.

Surgical Wounding

Wounding protocol was performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245–251 (1990)).Briefly, on the day of wounding, animals were anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal was shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area was dried with sterile gauze prior towounding. An 8 mm full-thickness wound was then created using a Keyestissue punch. Immediately following wounding, the surrounding skin wasgently stretched to eliminate wound expansion. The wounds were left openfor the duration of the experiment. Application of the treatment wasgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds were gently cleansed with sterilesaline and gauze sponges.

Wounds were visually examined and photographed at a fixed distance atthe day of surgery and at two day intervals thereafter. Wound closurewas determined by daily measurement on days 1–5 and on day 8. Woundswere measured horizontally and vertically using a calibrated Jamesoncaliper. Wounds were considered healed if granulation tissue was nolonger visible and the wound was covered by a continuous epithelium.

KGF-2 was administered using two different doses of KGF-2, one at 4 μgper wound per day for 8 days and the second at 40 μg per wound per dayfor 8 days in 50 μL of vehicle. Vehicle control groups received 50 μL ofvehicle solution.

Animals were euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin werethen harvested for histology and immunohistochemistry. Tissue specimenswere placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Experimental Design

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)were evaluated: 1) Vehicle placebo control, 2) KGF-2 4 μg/day and 3)KGF-2 40 μg/day. This study was designed as follows:

N Group Treatment N = 5 db+/db+ vehicle 50 μL N = 5 db+/+m vehicle 50 μLN = 5 db+/db+ KGF-2 4 μg/50 μL N = 5 db+/+m KGF-2 4 μg/50 μL N = 5db+/db+ KGF-2 40 μg/50 μL N = 5 db+/+m KGF-2 40 μg/50 μLMeasurement of Wound Area and Closure

Wound closure was analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction was then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 was 64 mm², the corresponding size of the dermalpunch. Calculations were made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]Histology

Specimens were fixed in 10% buffered formalin and paraffin embeddedblocks were sectioned perpendicular to the wound surface (5 μm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining was performed on cross-sections of bisected wounds. Histologicexamination of the wounds were used to assess whether the healingprocess and the morphologic appearance of the repaired skin was alteredby treatment with KGF-2. This assessment included verification of thepresence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)) (Table 1). A calibrated lensmicrometer was used by a blinded observer.

Immunohistochemistry

Re-Epithelialization

Tissue sections were stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin was used as a positive tissue control while non-immune IgGwas used as a negative control. Keratinocyte growth was determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Cell Proliferation Marker

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens wasdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer served as a positive tissue controland human brain tissue was used as a negative tissue control. Eachspecimen included a section with omission of the primary antibody andsubstitution with non-immune mouse IgG. Ranking of these sections wasbased on the extent of proliferation on a scale of 0–8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Statistical Analysis

Experimental data were analyzed using an unpaired t test. A p value of<0.05 was considered significant. The data were expressed as themean±SEM.

Results

Effect of KGF-2 on Wound Closure

Diabetic mice showed impaired healing compared to heterozygous normalmice. The dose of 4 μg of KGF-2 per site appeared to produce maximumresponse in diabetic and non-diabetic animals (FIG. 5, 6). These resultswere statistically significant (p=0.002 and p<0.0001) when compared withthe buffer control groups. Treatment with KGF-2 resulted in a finalaverage closure of 60.6% in the group receiving 4 μg/day and 34.5% inthe 40 μg/day group. Wounds in the buffer control group had only 3.8%closure by day 8. Repeated measurements of wounds on days 2–5post-wounding and on day 8 taken from the db+/db+mice treated with KGF-2demonstrated a significant improvement in the total wound area (sq. mm)by day 3 post-wounding when compared to the buffer control group. Thisimprovement continued and by the end of the experiment, statisticallysignificant results were observed (FIG. 7). Animals in the db/+m groupsreceiving KGF-2 also showed a greater reduction in the wound areacompared to the buffer control groups in repetitive measurements (FIG.8). These results confirmed a greater rate of wound closure in the KGF-2treated animals.

Effect of KGF-2 on Histologic Score

Histopathologic evaluation of KGF-2 in the diabetic (db+/db+) model onday 8 demonstrated a statistically significant improvement (p<0.0001) inthe wound score when compared with the buffer control. The pharmacologiceffects observed with both the 4 μg and the 40 μg doses of KGF-2 werenot significantly different from each other. The buffer control groupshowed minimal cell accumulation with no granulation tissue orepithelial travel while the 4 μg and 40 μg doses of KGF-2 (p<0.0001 &p=0.06 respectively) displayed epithelium covering the wound,neovascularization, granulation tissue formation and fibroblast andcollagen deposition (FIG. 9).

Histopathologic assessment of skin wounds was performed onhematoxylin-eosin stained samples. Scoring criteria included a scale of1–12, a score of one representing minimal cell accumulation with littleto no granulation and a score of 12 representing the abundant presenceof fibroblasts, collagen deposition and new epithelium covering thewound (Table 2).

TABLE 2 Scoring of Histology Sections Score Criteria 1–3 None to minimalcell accumulation. No granulation tissue or epithelial travel. 4–6 Thin,immature granulation that is dominated by inflammatory cells but has fewfibroblasts, capillaries or collagen deposition. Minimal epithelialmigration. 7–9 Moderately thick granulation tissue, can range from beingdominated by inflammatory cells to more fibroblasts and collagendeposition. Extensive neovascularization. Epithelium can range fromminimal to moderate migration. 10–12 Thick, vascular granulation tissuedominated by fibroblasts and extensive collagen deposition. Epitheliumpartially to completely covering the wound.

Evaluation of the non diabetic littermates, after both doses of KGF-2,showed no significant activity in comparison with the buffer controlgroup for all measurements evaluated (FIG. 10). The buffer control groupshowed immature granulation tissue, inflammatory cells, and capillaries.The mean score was higher than the diabetic group indicating impairedhealing in the diabetic (db+/db+) mice.

Effect of KGF-2 on Re-Epithelialization

Cytokeratine Immunostaining was used to determine the extent ofre-epithelialization. Scores were given based on degree of closure on ascale of 0 (no closure) to 8 (complete closure). In the groups receiving4 μg/day, there was a statistically significant improvement on there-epithelialization score when compared to the buffer control groupp<0.001 (FIG. 11). In this group, keratinocytes were observed localizedin the newly formed epidermis covering the wound. Both doses of KGF-2also exhibited mitotic figures in various stages. Assessment of thenon-diabetic groups at both doses of KGF-2 also significantly improvedreepithelialization ranking (p=0.006 and 0.01 respectively) (FIG. 12).

Effect of KGF-2 on Cell Proliferation

Proliferating cell nuclear antigen immunostaining demonstratedsignificant proliferation in both the 4 μg and 40 μg groups (FIG. 13).The non-diabetic group displayed similar results as both groupsreceiving both doses of KGF-2 showed higher significant scoring comparedto the buffer control group (FIG. 14). Epidermal proliferation wasobserved especially on the basal layer of the epidermis. In addition,high density PCNA-labeled cells were observed in the dermis, especiallyin the hair follicles.

Conclusion

The results demonstrate that KGF-2 specifically stimulates growth ofprimary epidermal keratinocytes. In addition, these experimentsdemonstrate that topically applied recombinant human KGF-2 markedlyaccelerates the rate of healing of full-thickness excisional dermalwounds in diabetic mice. Histologic assessment shows KGF-2 to inducekeratinocyte proliferation with epidermal thickening. This proliferationis localized in the basal layer of the epidermis as demonstrated byproliferating cell nuclear antigen (PCNA). At the level of the dermis,collagen deposition, fibroblast proliferation, and neo-vascularizationre-established the normal architecture of the skin.

The high density of PCNA-labeled cells on KGF-2 treated animals incontrast with the buffer group, which had fewer PCNA-labeled cells,indicates the stimulation of keratinocytes at the dermal-epidermallevel, fibroblasts and hair follicles. The enhancement of the healingprocess by KGF-2 was consistently observed in this experiment. Thiseffect was statistically significant in the parameters evaluated(percent re-epithelialization and wound closure). Importantly,PCNA-labeled keratinocytes were mainly observed at the lower-basal layerof the epidermis. The dermis showed normalized tissue with fibroblasts,collagen, and granulation tissue.

The activity observed in the non-diabetic animals indicates that KGF-2had significant pharmacologic response in the percentage of woundclosure at day 8, as well as during the course of the experiment, basedon daily measurements. Although the histopathologic evaluation was notsignificantly different when compared with the buffer control,keratinocyte growth and PCNA scores demonstrated significant effects.

In summary, these results demonstrated that KGF-2 shows significantactivity in both impaired and normal excisional wound models using thedb+/db+mouse model and therefore may be useful in the treatment ofwounds including surgical wounds, diabetic ulcers, venous stasis ulcers,burns, and other skin conditions.

EXAMPLE 8 KGF-2 Mediated Wound Healing in the Steroid-Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, S. M. Glucocorticoids andWound healing. In Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280–302 (1989); Wahl, S. M. et al., J. Immunol. 115: 476–481(1975); Werb, Z. et al., J. Exp. Med. 147:1684–1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert, R. H., et al., An. Intern. Med.37:701–705 (1952)), fibroblast proliferation, and collagen synthesis(Beck, L. S. et al., Growth Factors. 5: 295–304 (1991); Haynes, B. F.,et al., J. Clin. Invest. 61: 703–797 (1978)) and producing a transientreduction of circulating monocytes (Haynes, B. F., et al., J. Clin.Invest. 61: 703–797 (1978); Wahl, S. M. Glucocorticoids and woundhealing. In Antiinflammatory Steroid Action: Basic and Clinical Aspects.Academic Press. New York. pp. 280–302 (1989)). The systemicadministration of steroids to impaired wound healing is a well establishphenomenon in rats (Beck, L. S. et al., Growth Factors. 5: 295–304(1991); Haynes, B. F., et al., J. Clin. Invest. 61: 703–797 (1978);Wahl, S. M. Glucocorticoids and wound healing. In AntiinflammatorySteroid Action: Basic and Clinical Aspects. Academic Press. New York.pp. 280–302 (1989); Pierce, G. F., et al., Proc. Natl. Acad. Sci. USA.86: 2229–2233 (1989)).

To demonstrate that KGF-2 would accelerate the healing process, theeffects of multiple topical applications of KGF-2 on full thicknessexcisional skin wounds in rats in which healing has been impaired by thesystemic administration of methylprednisolone was assesed. In vitrostudies have demonstrated that KGF-2 specifically stimulates growth ofprimary human epidermal keratinocytes. This example demonstrates thattopically applied recombinant human KGF-2 accelerates the rate ofhealing of full-thickness excisional skin wounds in rats by measuringthe wound gap with a calibrated Jameson caliper and by histomorphometryand immunohistochemistry. Histologic assessment demonstrates that KGF-2accelerates re-epithelialization and subsequently, wound repair.

Animals

Young adult male Sprague Dawley rats weighing 250–300 g (Charles RiverLaboratories) were used in this example. The animals were purchased at 8weeks of age and were 9 weeks old at the beginning of the study. Thehealing response of rats was impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals were individually housed and received food and waterad libitum. All manipulations were performed using aseptic techniques.This study was conducted according to the rules and guidelines of HumanGenome Sciences, Inc. Institutional Animal Care and Use Committee andthe Guidelines for the Care and Use of Laboratory Animals.

KGF-2

Recombinant human KGF-2 was over-expressed and purified frompQE60-Cys37, an E. coli expression vector system (pQE-9, Qiagen). Theprotein expressed from this construct is the KGF-2 from Cysteine atposition 37 to Serine at position 208 with a 6×(His) tag attached to theN-terminus of the protein (FIG. 15) (SEQ ID NOS:29–30). Fractionscontaining greater than 95% pure recombinant materials were used for theexperiment. KGF-2 was formulated in a vehicle containing 1×PBS. Thefinal concentrations were 20 μg/mL and 80 μg/mL of stock solution.Dilutions were made from stock solution using the same vehicle.

KGF-2 Δ28 was over-expressed and purified from an E. coli expressionvector system. Fractions containing greater than 95% pure recombinantmaterials were used for the experiment. KGF-2 was formulated in avehicle containing 1×PBS. The final concentrations were 20 μg/mL and 80μg/mL of stock solution. Dilutions were made from stock solution usingthe same vehicle.

Surgical Wounding

The wounding protocol was followed according to Example 7, above. On theday of wounding, animals were anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal was shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area was dried with sterile gauze priorto wounding. An 8 mm full-thickness wound was created using a Keyestissue punch. The wounds were left open for the duration of theexperiment. Applications of the testing materials were given topicallyonce a day for 7 consecutive days commencing on the day of wounding andsubsequent to methylprednisolone administration. Prior to treatment,wounds were gently cleansed with sterile saline and gauze sponges.

Wounds were visually examined and photographed at a fixed distance atthe day of wounding and at the end of treatment. Wound closure wasdetermined by daily measurement on days 1–5 and on day 8 for Figure.Wounds were measured horizontally and vertically using a calibratedJameson caliper. Wounds were considered healed if granulation tissue wasno longer visible and the wound was covered by a continuous epithelium.

A dose response was performed using two different doses of KGF-2, one at1 μg per wound per day and the second at 4 μg per wound per day for 5days in 50 μL of vehicle. Vehicle control groups received 50μL of 1×PBS.

Animals were euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin werethen harvested for histology. Tissue specimens were placed in 10%neutral buffered formalin in tissue cassettes between biopsy sponges forfurther processing.

Experimental Design

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) were evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) KGF-2 1 μg/day and 4) KGF-2 4 μg/day. This study was designedas follows:

n Group Treatment Glucocorticoid-Treated N = 5 Untreated — N = 5 Vehicle50 μL N = 5 KGF-2 (1 μg) 50 μL N = 5 KGF-2 (4 μg) 50 μL WithoutGlucocorticoid N = 5 Untreated — N = 5 Vehicle 50 μL N = 5 KGF-2 (1 μg)50 μL N = 5 KGF-2 (4 μg) 50 μLMeasurement of Wound Area and Closure

Wound closure was analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure wasthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1was 64 mm², the corresponding size of the dermal punch. Calculationswere made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]Histology

Specimens were fixed in 10% buffered formalin and paraffin embeddedblocks were sectioned perpendicular to the wound surface (5 μm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining wasperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allowed us to assess whether the healing process and themorphologic appearance of the repaired skin was improved by treatmentwith KGF-2. A calibrated lens micrometer was used by a blinded observerto determine the distance of the wound gap.

Statistical Analysis

Experimental data were analyzed using an unpaired t test. A p value of<0.05 was considered significant. The data was expressed as themean±SEM.

Results

A comparison of the wound closure of the untreated control groups withand without methylprednisolone demonstrates thatmethylprednisolone-treated rats have significant impairment of woundhealing at 8 days post-wounding compared with normal rats. The totalwound area measured 58.4 mm² in the methylprednisolone injected groupand 22.4 mm² in the group not receiving glucocorticoid (FIG. 16).

Effect of KGF-2 on Wound Closure

Systemic administration of methylprednisolone in rats at the time ofwounding delayed wound closure (p=0.002) of normal rats. Wound closuremeasurements of the methlyprednisolone-impaired groups at the end of theexperiment on day 8 demonstrated that wound closure with KGF-2 wassignificantly greater statistically (1 μg p=0.002 & 4 μg p=0.005) whencompared with the untreated group (FIG. 16). Percentage wound closurewas 60.2% in the group receiveing 1 μg KGF-2 (p=0.002) and 73% in thegroup receiving 4 μg KGF-2 (p=0.0008). In contrast, the wound closure ofuntreated group was 12.5% and the vehicle placebo group was 28.6% (FIG.17).

Longitudinal analysis of wound closure in the glucocorticoid groups fromday 1 to 8 shows a significant reduction of wound size from day 3 to 8postwounding in both doses of KGF-2 in the treated groups (FIG. 18).

The results demonstrate that the group treated with the 4 μg KGF-2 hadstatistically significant (p=0.05) accelerated wound closure comparedwith the untreated group (FIG. 19A). Although it is difficult to assessthe ability of a protein or other compounds to accelerate wound healingin normal animal (due to rapid recovery), nonetheless, KGF-2 was shownto accelerate wound healing in this model.

Histopathologic Evaluation of KGF-2 Treated Wounds

Histomorphometry of the wound gap indicated a reduction in the wounddistance of the KGF-2 treated group. The wound gap observed for theuntreated group was 5336 μ while the group treated with 1 μg KGF-2 had awound gap reduction to 2972μ; and the group treated with 4 μg KGF-2(p=0.04) had a wound gap reduction to 3086μ (FIG. 20).

Effects of KGF-2 Δ28 in Wound Healing

Evaluation of KGF-2 Δ28 and PDGF-BB in wound healing in themethylprednisolone impared rat model was also examined. The experimentwas carried out the same as for the KGF-2 protein above, except that theKGF-2 Δ28 protein is not His tagged and wound healing was measured ondays 2,4,6,8, and 10. The buffer vehicle for the proteins was 40 mMNaOAc and 150 mM NaCl, pH6.5 for all but the “E2” preparation of thefull length KGF-2. The buffer vehicle for the “E2” KGF-2 preparation was20 mM NaOAc and 400 mM NaCl, pH6.4.

The results shown in FIG. 19B demonstrate that KGF-2 Δ28 hasstatistically significant accelerated wound closure compared with theuntreated group and has reversed the effects of methylprednisolone onwound healing.

Conclusions

This example demonstrates that KGF-2 reversed the effects ofmethylprednisolone on wound healing. The exogenous application of growthfactors may accelerate granulation tissue formation by drawinginflammatory cells into the wound. Similar activity was also observed inanimals not receiving methylprednisolone indicating that KGF-2 hadsignificant pharmacologic response in the percentage of wound closure atday 5 based on daily measurements. The glucocorticoid-impaired woundhealing model in rats was shown to be a suitable and reproducible modelfor measuring efficacy of KGF-2 and other compounds in the wound healingarea.

In summary, the results demonstrate that KGF-2 shows significantactivity in both glucocorticoid impaired and in normal excisional woundmodels. Therefore, KGF-2 may be clinically useful in stimulating woundhealing including surgical wounds, diabetic ulcers, venous stasisulcers, burns, and other abnormal wound healing conditions such asuremia, malnutrition, vitamin deficiencies and systemic treatment withsteroids and antineoplastic drugs.

EXAMPLE 9 Tissue Distribution of KGF-2 mRNA Expression

Northern blot analysis is carried out to examine the levels ofexpression of the gene encoding the KGF-2 protein in human tissues,using methods described by, among others, Sambrook et al., cited above.A probe corresponding to the entire open reading frame of KGF-2 of thepresent invention (SEQ ID NO:1) was obtained by PCR and was labeled with³²P using the rediprime™ DNA labeling system (Amersham Life Science),according to manufacturer's instructions. After labelling, the probe waspurified using a CHROMA SPIN-100™ column (Clontech Laboratories, Inc.),according to manufacturer's protocol number PT1200-1. The purifiedlabelled probe was then used to examine various human tissues for theexpression of the gene encoding KGF-2.

Multiple Tissue Northern (MTN) blots containing poly A RNA from varioushuman tissues (H) or human immune system tissues (IM) were obtained fromClontech and were examined with labelled probe using ExpressHyb™Hybridization Solution (Clontech) according to manufacturer's protocolnumber PT1190-1. Following hybridization and washing, the blots aremounted and exposed to film at −70° C. overnight, and films developedaccording to standard procedures.

A major mRNA species of approximately 4.2 kb was observed in most humantissues. The KGF-2 mRNA was relatively abundant in heart, pancreas,placenta and ovary. A minor mRNA species of about 5.2 kb was alsoobserved ubiquitously. The identity of this 5.2 kb mRNA species was notclear. It is possible that the 5.2 kb transcript encodes analternatively spliced form of KGF-2 or a third member of the KGF family.The KGF-2 cDNA was 4.1 kb, consistent with the size of the mRNA of 4.2kb.

EXAMPLE 10 Keratinocyte Proliferation Assays

Dermal keratinocytes are cells in the epidermis of the skin. The growthand spreading of keratinocytes in the skin is an important process inwound healing. A proliferation assay of keratinocyte is therefore avaluable indicator of protein activities in stimulating keratinocytegrowth and consequently, wound healing.

Keratinocytes are, however, difficult to grow in vitro. Few keratinocytecell lines exist. These cell lines have different cellular and geneticdefects. In order to avoid complications of this assay by cellulardefects such as loss of key growth factor receptors or dependence of keygrowth factors for growth, primary dermal keratinocytes are chosen forthis assay. These primary keratinocytes are obtained from Clonetics,Inc. (San Diego, Calif.).

Keratinocyte proliferation assay with AlamarBlue

AlamarBlue is a viable blue dye that is metabolized by the mitochondriawhen added to the culture media. The dye then turns red in tissueculture supernatants. The amounts of the red dye may be directlyquantitated by reading difference in optical densities between 570 nmand 600 nm. This reading reflects cellular activities and cell number.

Normal primary dermal keratinocytes (CC-0255, NHEK-Neo pooled) arepurchased from Clonetics, Inc. These cells are passage 2. Keratinocytesare grown in complete keratinocyte growth media (CC-3001, KGM;Clonetics, Inc.) until they reach 80% confluency. The cells aretrypsinized according to the manufacturer's specification. Briefly,cells were washed twice with Hank's balanced salt solution. 2–3 ml oftrypsin was added to cells for about 3–5 min at room temperature.Trypsin neutralization solution was added and cells were collected.Cells are spun at 600×g for 5 min at room temperature and plated intonew flasks at 3,000 cells per square centimeter using pre-warmed media.

For the proliferation assay, plate 1,000–2,000 keratinocytes per well ofthe Corning flat bottom 96-well plates in complete media except for theoutermost rows. Fill the outer wells with 200 μl of sterile water. Thishelps to keep temperature and moisture fluctuations of the wells to theminimum. Grow cells overnight at 37° C. with 5% CO₂. Wash cells twicewith keratinocyte basal media (CC-3101, KBM, Clonetics, Inc.) and add100 μl of KBM into each well. Incubate for 24 hours. Dilute growthfactors in KBM in serial dilution and add 100 μl to each well. Use KGMas a positive control and KBM as a negative control. Six wells are usedfor each concentration point. Incubate for two to three days. At the endof incubation, wash cells once with KBM and add 100 μl of KBM with 10%v/v alamarBlue pre-mixed in the media. Incubate for 6 to 16 hours untilmedia color starts to turn red in the KGM positive control. Measure O.D.570 nm minus O.D. 600 nm by directly placing plates in the plate reader.

Results

Stimulation of Keratinocyte Proliferation by KGF-2

To demonstrate that KGF-2 (i.e., starting at amino acid Cys37 asdescribed in Examples 7 and 8 above) and N-terminal deletion mutantsKGF-2 Δ33 and KGF-2 Δ28 were active in stimulating epidermalkeratinocyte growth, normal primary human epidermal keratinocytes wereincubated with the E. coli-expressed and purified KGF-2 protein (batchnumber E3)(SEQ ID NO: 2), KGF-2 Δ33 (batch number E1) and KGF-2 Δ28(batch number E2). The KGF-2 protein stimulated the growth of epidermalkeratinocytes with an EC50 of approximately 5 ng/ml, equivalent to thatof FGF7/KGF-1 (FIG. 21A). In contrast, other FGF's such as FGF-1 andFGF-2 did not stimulate the growth of primary keratinocytes. The EC50for KGF-2 Δ33 was 0.2 ng/ml and that for KGF-2 Δ28 2 ng/ml (See FIGS.21B and C). Thus, KGF-2 appeared to be as potent as FGF7/KGF instimulating the proliferation of primary epidermal keratinocytes.However, KGF-2 Δ33 is more potent in stimulating keratinocyteproliferation than the “Cys (37)” KGF-2 described in Examples 7 and 8above and the KGF-2 Δ28.

Scarring of wound tissues involves hyperproliferation of dermalfibroblasts. To determine whether the stimulatory effects of KGF-2 wasspecific for keratinocytes but not for fibroblasts, mouse Balb.c.3T3fibroblsts and human lung fibroblasts were tested. Niether types offibroblasts responded to KGF-2 in proliferation assays. Therefore, KGF-2appeared to be a mitogen specific for epidermal keratinocytes but notmesenchymal cells such as fibroblasts. This suggested that thelikelyhood of KGF-2 causing scarring of the wound tissues was low.

EXAMPLE 11 A. Mitogenic Effects of KGF-2 on Cells Transfected withSpecific FGF Receptors

To determine which FGF receptor isoform(s) mediate the proliferativeeffects of KGF-2, the effects of KGF-2 on cells expressing specific FGFreceptor isoforms were tested according to the method described bySantos-Ocampo et al. J. Biol. Chem. 271:1726–1731 (1996). FGF7/KGF wasknown to induce mitogenesis of epithelial cells by binding to andspecifically activating the FGFR2iiib form (Miki et al. Science251:72–75 (1991)). Therefore, the proliferative effects of KGF-2 inmitogensis assays were tested using cells expressing one of thefollowing FGF receptor isoforms: FGFR1iiib, FGFR2iiib, FGFR3iiib, andFGFR4.

Mitogensis Assay of Cells Expressing FGF Receptors

Thymidine incorporation of BaF3 cells expressing specific FGF receptorswere performed as described by Santos-Ocampo et al. J. Biol. Chem.271:1726–1731 (1996). Briefly, BaF3 cells expressing specific FGFreceptors were washed and resuspended in Dulbecco's modified Eagle'smedium, 10% neonatal bovine serum, L-glutanime. Approximately 22,500cells were plated per well in a 96-well assay plate in media containing2 μg/ml Heparin. Test reagents were added to each well for a totalvolume of 200 μl per well. The cells were incubated for 2 days at 37° C.To each well, 1 μCi of ³H-thymidine was then added in a volume of 50 μl.Cells were harvested after 4–5 hours by filteration through glass fiberpaper. Incorporated ³H-thymidine was counted on a Wallac beta platescintillaion counter.

Results

The results revealed that KGF-2 protein (Thr (36)—Ser (208) of FIGS.1A–1C (SEQ ID NO:2) with a N-terminal Met added thereto) stronglystimulated the proliferation of Baf3 cells expressing the KGF receptor,FGFR2iiib isoform, as indicated by ³H-thymidine incorporation (FIG.22A). Interestingly, a slight stimulatory effect of KGF-2 on theproliferation of Baf3 cells expressing the FGFR1iiib isoform wasobserved. KGF-2 did not have any effects on cells expressing theFGFR3iiib or the FGFR4 forms of the receptor.

FGF7/KGF stimulated the proliferation of cells expressing the KGFreceptor, FGFR2iiib but not FGFR1iiib isoform. The difference betweenKGF-2 and FGF7/KGF was intriguing. In the control experiments, aFGFstimulated its receptors, FGFR1iiib and iiic and bFGF stimulated itsreceptor FGFR2iiic. Thus, these results suggested that KGF-2 binds toFGFR2iiib isoform and stimulates mitogenesis. In contrast to FGF7/KGF,KGF-2 also binds FGFR1iiib isoform and stimulates mitogenesis.

B. Mitogenic effects of KGF-2 Δ33 on Cells Transfected with Specific FGFReceptors

As demonstrated above FGFs or KGF-1 and -2 both bind to and activate theFGF 2iiib receptor (FGFR 2iiib). The proliferative effects of KGF-2 Δ33in mitogenesis assays were tested using cells expressing one of thefollowing FGF receptor isoforms: FGFR2iiib or FGFR2iiic (the 2iiicreceptor-transfected cells are included as a negative control).

The experiments were performed as above in part A of this example.Briefly, BaF3 cells were grown in RPMI containing 10% bovine calf serum(BCS—not fetal serum), 10% conditioned medium from cultures of WEHI3cells (grown in RPMI containing 5% BCS), 50 nM β-mercaptoethanol, L-Glu(2% of a 100× stock) and pen/strep (1% of a 100× stock).

For the assay, BaF3 cells were rinsed twice in RPMI medium containing10% BCS and 1 μg/ml heparin. BaF3 cells (22,000/well) were plated in a96-well plate in 150 μl of RPMI medium containing 10% BCS and 1 μg/mlheparin. Acidic FGF, basic FGF, KGF-1 (HG15400) or KGF-2 proteins(HG03400, 03401, 03410 or 03411) were added at concentrations fromapproximately 0 to 10 nM. The cells were incubated in a final volume of200 μl for 48 hours at 37° C. All assays were done in triplicate.Tritiated thymidine (0.5 μCi) was added to each well for 4 hours at 37°C. and the cells were then harvested by filtration through a glass fiberfilter. The total amount of radioactivity incorporated was thendetermined by liquid scintillation counting. The following positivecontrols were used: basic FGF and acidic FGF for FGFR2iiic cells; acidicFGF and KGF-1 for FGFR2iiib cells. The following negative controls wereused: Basal medium (RPMI medium containing 10% BCS and 1 μg/ml heparin).

Results:

The results revealed that KGF-2 (Thr (36)—Ser (208) with N-terminal Metadded), KGF-2 Δ33 and KGF-2 Δ28 proteins strongly stimulated theproliferation of BaF3 cells expressing the KGF receptor, FGFR2iiibisoform, as indicated by ³H-thymidine incorporation (FIGS. 22A–C). TheKGF-2 proteins did not have any effects on cells expressing theFGFR2iiic forms of the receptor. These results suggested that KGF-2proteins bind to FGFR2iiib isoform and stimulates mitogenesis. Inaddition, it appears that KGF-2 Δ33 was able to stimulate theproliferation of the BaF3 cells better than the KGF-2 (Thr (36)—Ser(208)).

EXAMPLE 12 A. Construction of E. coli Optimized Full Length KGF-2

In order to increase expression levels of full length KGF-2 in an E.coli expression system, the codons of the amino terminal portion of thegene were optimized to highly used E. coli codons. For the synthesis ofthe optimized region of KGF-2, a series of six oligonucleotides weresynthesized: numbers 1–6 (sequences set forth below). These overlappingoligos were used in a PCR reaction for seven rounds at the followingconditions:

Denaturation 95 degrees 20 seconds Annealing 58 degrees 20 secondsExtension 72 degrees 60 seconds

A second PCR reaction was set up using 1 μl of the first PCR reactionwith KFG-2 synthetic primer 6 as the 3′ primer and KGF-2 synthetic 5′BamHI as the 5′ primer using the same conditions as described above for25 cycles. The product produced by this final reaction was restrictedwith AvaII and BamHI. The KGF-2 construct of Example 1 was restrictedwith AvaII and HindIII and the fragment was isolated. These twofragments were cloned into pQE-9 restricted with BamHI and HindIII in athree fragment ligation.

Primers used for constructing the optimized synthetic KGF-2 1/208:

KGF-2 Synthetic Primer 1:ATGTGGAAATGGATACTGACCCACTGCGCTTCTGCTTTCCCGCACCTG (SEQ ID NO: 31)CCGGGTTGCTGCTGCTGCTGCTTCCTGCTGCTGTTC KGF-2 Synthetic Primer 2:CCGGAGAAACCATGTCCTGACCCAGAGCCTGGCAGGTAACCGGAACA (SEQ ID NO: 32)GAAGAAACCAGGAACAGCAGCAGGAAGCAGCAGCA KGF-2 Synthetic Primer 3:GGGTCAGGACATGGTTTCTCCGGAAGCTACCAACTCTTCTTCTTCTTCT (SEQ ID NO: 33)TTCTCTTCTCCGTCTTCTGCTGGTCGTCACG KGF-2 Synthetic Primer 4:GGTGAAAGAGAACAGTTTACGCCAACGAACGTCACCCTGCAGGTGGT (SEQ ID NO: 34)TGTAAGAACGAACGTGACGACCAGCAGAAGACGG KGF-2 Synthetic Primer 5:CGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAA (SEQ ID NO: 35)AAAAACGGTAAAGTTTCTGGGACCAAA KGF-2 Synthetic Primer 6:TTTGGTCCCAGAAACTTTACCGTTTTTTTCGATTTTCAG (SEQ ID NO: 36) KGF-2 Synthetic5′ BamHI AAAGGATCCATGTGGAAATGGATACTGACCCACTGC (SEQ ID NO: 37)

The resulting clone is shown in FIG. 23 (SEQ ID NOS: 38 and 39).

B. Construction of E. coli Optimized Mature KGF-2

In order to further increase expression levels of the mature form ofKGF-2 in an E. coli expression system, the codons of the amino terminalportion of the gene were optimized to highly used E. coli codons. Tocorrespond with the mature form of KGF-1, a truncated form of KGF-2 wasconstructed starting at threonine 36. E. coli synthetic KGF-2 fromExample 12A was used as a template in a PCR reaction using BspHI 5′KGF-2 as the 5′ primer (sequence given below) and HindIII 3′ KGF-2 asthe 3′ primer (sequence given below). Amplification was performed usingstandard conditions as given above in Example 12A for 25 cycles. Theresulting product was restricted with BspHI and HindII and cloned intothe E. coli expression vector pQE60 digested with NcoI and HindIII.

BspHI 5′ KGF-2 Primer: TTTCATGACTTGTCAAGCTCTGGGTCAA (SEQ ID NO:40)GATATGGTTC HindIII 3′ KGF-2 Primer: GCCCAAGCTTCCACAAACGTTGCCTTCC (SEQ IDNO:41)

The resulting clone is shown in FIG. 24A (SEQ ID NO:42 and 43).

C. Construction of an Alternate E. coli Optimized Mature KGF-2

In order to further increase expression levels of the mature form ofKGF-2 in an E. coli expression system, the codons of 53 amino acids atthe amino terminal portion of the E. coli optimized gene were changed toalternate highly used E. coli codons. For the synthesis of the optimizedregion of KGF-2, a series of six oligonucleotides were synthesized:numbers 18062, 18061, 18058, 18064, 18059, and 18063 (sequences setforth below). These overlapping oligos were used in a PCR reaction forseven rounds at the following conditions:

Denaturation 95 degrees 20 seconds Annealing 58 degrees 20 secondsExtension 72 degrees 60 seconds

Following the seven rounds of synthesis, a 5′ primer to this region,18169 and a 3′ primer to this entire region, 18060, were added to a PCRreaction, containing 1 microliter from the initial reaction of the sixoligonucleotides. This product was amplified for 30 rounds using thefollowing conditions:

Denaturation 95 degrees 20 seconds Annealing 55 degrees 20 secondsExtension 72 degrees 60 seconds

A second PCR reaction was set up to amplify the 3′ region of the geneusing primers 18066 and 18065 under the same conditions as describedabove for 25 rounds. The resulting products were separated on an agarosegel. Gel slices containing the product were diluted in 10 mM Tris, 1 mMEDTA, pH 7.5 One microliter each from each of diluted gel slices wereused in an additional PCR reaction using primer 18169 as the 5′ primer,and primer 18065 as the 3′ primer. The product was amplified for 25cycles using the same conditions as above. The product produced by thisfinal reaction was and restricted with EcoRI and HindIII, and clonedinto pQE60, which was also cut with EcoRI and HindIII (pQE6 now).

Sequences of the 5′ Synthetic Primers:

18169 KGF2 5′ EcoRI/RBS: TCAGTGAATTCATTAAAGAGGAGAAATTAATCATGACTTGCCAGG[SEQ ID NO:44] 18062 KGF2 synth new R1 sense:TCATGACTTGCCAGGCACTGGGTCAAGACATGGTTTCCCCGGAAGCTA [SEQ ID NO:45] 18061KGF2 synth R2 sense: GCTTCAGCAGCCCATCTAGCGCAGGTCGTCACGTTCGCTCTTACAACC[SEQ ID NO:46] 18058 KGF2 Synth R3 sense:GTTCGTTGGCGCAAACTGTTCAGCTTTACCAAGTACTTCCTGAAAATC [SEQ ID NO:47] 18066KGF 2 20 bp Ava II sense: TCGAAAAAAACGGTAAAGTTTCTGGGAC [SEQ ID NO:48]18064 KGF2 synth F1 antisense:GATGGGCTGCTGAAGCTAGAGCTGGAGCTGTTGGTAGCTTCCGGGGAA [SEQ ID NO:49] 18059KGF2 Synth F2 antisense: AACAGTTTGCGCCAACGAACATCACCCTGTAAGTGGTTGTAAGAG[SEQ ID NO:50] 18063 KGF2 Synth F3 antisense:TTCTTGGTCCCAGAAACTTTACCGTTTTTTTCGATTTTCAGGAAGTA [SEQ ID NO:51] 18060 KGF2 Ava II antisense: TTCTTGGTCCCAGAAACTTTACCG [SEQ ID NO:52] 18065 KGF2HindIII 3′ Stop: AGATCAGGCTTCTATTATTATGAGTGTACCACCATTGGAAGAAAG [SEQ IDNO:53]

The sequence of the synthetic KGF-2 gene and it corresponding amino acidis shown in FIG. 24B (SEQ ID NO: 54 and 55)

EXAMPLE 13 Construction of KGF-2 Deletion Mutants

Deletion mutants were constructed from the 5′ terminus and 3′ terminusof KGF-2 gene using the optimized KGF-2 construct from Example 12A as atemplate. The deletions were selected based on regions of the gene thatmight negatively affect expression in E. coli. For the 5′ deletion theprimers listed below were used as the 5′ primer. These primers containthe indicated restriction site and an ATG to code for the initiatormethionine. The KGF-2 (FGF-12) 208 amino acid 3′ HindIII primer was usedfor the 3′ primer. PCR amplification for 25 rounds was performed usingstandard conditions as set forth in Example 12. The products for theKGF-236aa/208aa deletion mutant were restricted BspHI for the 5′ siteand HindIII for the 3′ site and cloned into the pQE60 which has beendigested with BspHI and HindIII. All other products were restricted withNcoI for the 5′ restriction enzyme and HindIII for the 3′ site, andcloned into the pQE60 which had been digested with NcoI and HindIII. ForKGF-2 (FGF-12), 36aa/153aa and 128aa 3′ HindIII was used as the 3′primer with FGF-12 36aa/208aa as the 5′ primer. For FGF-12 62aa/153aa,128aa 3′ HindIII was used as the 3′ primer with FGF-12 62aa/208aa as the5′ primer. The nomenclature of the resulting clones indicates the firstand last amino acid of the polypeptide that results from the deletion.For example, KGF-2 36aa/153aa indicates that the first amino acid of thedeletion mutant is amino acid 36 and the last amino acid is amino acid153 of KGF-2. Further, as indicated in FIGS. 25–33, each mutant hasN-terminal Met added thereto.

Sequences of the Deletion Primers:

FGF12 36aa/208aa: 5′ BsphI GGACCCTCATGACCTGCCAGGCTCTGGGTCAGGAC [SEQ IDNO:56] FGF12 63aa/208aa: 5′ NcoI GGACAGCCATGGCTGGTCGTCACGTTCG [SEQ IDNO:57] FGF12 77aa/208aa: 5′ NcoI GGACAGCCATGGTTCGTTGGCGTAAACTG [SEQ IDNO:58] FGF12 93aa/208aa: 5′ NcoI GGACAGCCATGGAAAAAAACGGTAAAGTTTC [SEQ IDNO:59] FGF12 104aa/208aa: 5′ NcoI GGACCCCCATGGAGAACTGCCCGTAGAGC [SEQ IDNO:60] FGF12 123aa/208aa: 5′ NcoI GGACCCCCATGGTCAAAGCCATTAACAGCAAC [SEQID NO:61] FGF12 138aa/208aa: 5′ NcoI GGACCCCCATGGGGAAACTCTATGGCTCAAAAG[SEQ ID NO:62] FGF12 3′ HindIII: (Used for all above deletion clones)CTGCCCAAGCTTAYTTATGAGTGTACCACCATTGGAAG [SEQ ID NO:63] FGF12 36aa/153aa:5′ BsphI (as above) 3′HindIII CTGCCCAAGCTTATTACTTCAGCTTACAGTCATTGT [SEQID NO:64]FGF12 63aa/153aa:5′ NcoI and 3′ HindIII, as above

The sequences for the resulting deletion mutations are set forth inFIGS. 25–33. [SEQ ID NOS:65–82].

When expressing KGF-2 Δ28 (amino acids 63–208) in E. coli, a proteaseinhibitor, such as Guanidine Hydrochloride (Gu-HCl), is used preventdegradation of the protein. For example, the E. coli paste isresuspended in 50 mM Tris-Acetate, 10 mM EDTA-NA₂, pH 7.7±0.2 followedby lysis. The lysed suspension is treated with an equal volume of 1.0 MGu-HCl solution and gently stirred for 2–4 hours at 2–8° C. Thesuspension is then centrifuges and filtered before loading onto thefirst column for purification. Initial purification takes place on aSP-Sepharose FF column wherein the bound KGF-2 is eluted with a saltgradient. The resulting SP-Sepharose elution pool is diluted and 0.2 μmfiltered and loaded onto a Fractogel COO⁻(S) column. Elution is carriedout through a salt gradient and the elution pool is diafiltered andconcentrated into a buffer.

EXAMPLE 14 Construction of Cysteine Mutants of KGF-2

Construction of C-37 mutation primers 5457 5′ BspHI and 5258 173aa 3′HindIII were used to amplify the KGF-2 (FGF-12) template from Example 12A. Primer 5457 5′ BspHI changes cysteine 37 to a serine. Amplificationwas done using the standard conditions outlined above in Example 12A for25 cycles. The resulting product was restricted with BspHI and HindIIIand cloned into E. coli expression vector pQE60, digested with BspHI andHindIII. (FIG. 34) [SEQ ID NO:83]

For mutation of Cysteine 106 to serine, two PCR reactions were set upfor oligonucleotide site directed mutagenesis of this cysteine. In onereaction, 5453 BspHI was used as the 5′ primer, and 5455 was used as the3′ primer in the reaction. In a second reaction, 5456 was used as the 5′primer, and 5258 HindIII was used as the 3′ primer. The reactions wereamplified for 25 rounds under standard conditions as set forth inExample 12. One microliter from each of these PCR reactions was used astemplate in a subsequent reaction using, as a 5′ primer, 5453 BspHI, andas a 3′ primer, 5258 HindIII. Amplification for 25 rounds was performedusing standard conditions as set forth in Example 12. The resultingproduct was restricted with BspHI and HindIII and cloned into the E.coli expression vector pQE60, which was restricted with NcoI andHindIII.

Two PCR reactions were required to make the C-37/C-106 mutant. Primers5457 BspHI and 5455 were used to create the 5′ region of the mutantcontaining cysteine 37 to serine substitution, and primer 5456 and 5258HindIII were used to create the 3′ region of the mutant containingcysteine 106 to serine substitution. In the second reaction, the 5457BspHI primer was used as the 5′ primer and the 5258 HindIII primer wasused as the 3′ primer to create the C-37/C-106 mutant using 1 μl fromeach of the initial reactions together as the template. This PCR productwas restricted with BspHI and HindIII, and cloned into pQE60 that hadbeen restricted with NcoI and HindIII. The resulting clone is shown inFIG. 35 (SEQ ID NO:84)

Sequences of the Cysteine Mutant Primers:

5457 BspHI: GGACCCTCATGACCTCTCAGGCTCTGGGT (SEQ ID NO:85) 5456:AAGGAGAACTCTCCGTACAGC (SEQ ID NO:86) 5455: GCTGTACGGTCTGTTCTCCTT (SEQ IDNO:87) 5453 BspHI: GGACCCTCATGACCTGCCAGGCTCTGGGTCAGGAC (SEQ ID NO:88)5258 HindIII: CTGCCCAAGCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID NO:89)

EXAMPLE 15 Production and Purification of KGF-2 (FGF-12)

The DNA sequence encoding the optimized mature protein described inExample 12B (i.e., amino acids T36 through S208 of KGF-2) was clonedinto plasmid pQE-9 (Qiagen). E. coli (M15/rep4;Qiagen) were grown tostationary phase overnight at 37° C. in LB containing 100 μg/mlAmpicillin and 25 μg/ml Kanamycin. This culture was used to innoculatefresh LB media containing 100 μg/ml Ampicillin and 25 μg/ml Kanamycin ata dilution of 1:50. The cells were grown at 37° C. to an O.D.₅₉₅ of 0.7,induced by the addition of isopropyl 1-thio-b-D-galactopyranoside (IPTG)to a final concentration of 1 mM. After 3–4 hours, the cells wereharvested by centrifugation, and resuspended in a buffer containing 60mM NaPO₄ and 360 mM NaCl at a ratio of 5 volumes of buffer: 1 volume ofcell paste. After disruption in a Mautin Gaulin, the extract wasadjusted to pH to 8.0 by the addition of NaOH and clarified bycentrifugation.

The clarified soluble extract was applied to a Poros HS-50 column(2.0×10.0 cm; PerSeptive Biosystems, Inc.) and bound proteinsstep-eluted with 50 mM NaPO₄ pH 8.0 containing 0.5M, 1.0M and 1.5M NaCl.The KGF-2 eluted in the 1.5M salt fraction which was then dilutedfive-fold with 50 mM NaPO₄ pH 6.5 to a final salt concentration of 300mM. This KGF-2 containing fraction was then passed sequentially over aPoros HQ-20 column (2.0×7.0 cm; PerSeptive Biosystems, Inc.) and thenbound to a Poros CM-20 column (2.0×9.0 cm; PerSeptive Biosystems, Inc.).KGF-2 (FGF-12)-containing fractions that eluted at about 500 mM to about750 mM NaCl were pooled, diluted and re-applied to an CM-20 column toconcentrate. Finally, the protein was seperated on a gel filtrationcolumn (S-75; Pharmacia) in 40 mM NaOAC pH6.5; 150 mM NaCl (Batch E-5)Alternatively, the gel filtration column was run in Phosphate BufferedSaline (PBS, Batch E-4). KGF-2 containing fractions were pooled andprotein concentration determined by Bio-Rad Protein Assay. Proteins werejudged to be>90% pure by SDS-PAGE. Finally, endotoxin levels determinedby Limulus Amebocyte Lysate Assay (Cape Cod Associates) were found tobe<lEu/mg. Proteins prepared in this way were able to bind heparin whichis a hallmark of FGF family members.

EXAMPLE 16 A. Construction of N-Terminal Deletion Mutant KGF-2 Δ33

To increase the level of expression of KGF2 in E. coli, and to enhancethe solubilty and stability properties of E. coli expressed KGF2, adeletion variant KGF-2 Δ33 (KGF-2 aa 69–208) (SEQ ID NO:96) whichremoves the first 68 amino acids of the pre-processed KGF2 wasgenerated. The rationale for creating this deletion variant was based onthe following observations. Firstly, mature KGF2 (KGF-2 aa 36–208)contains an uneven number (three) of cysteine residues which can lead toaggregation due to intra-molecular disulphide bridge formation. TheKGFΔ33 deletion variant contains only two cysteine residues, whichreduces the potential for intra-molecular disulphide bridge formationand subsequent aggregation. A decrease in aggregation should lead to anincrease in the yield of active KGF2 protein. Secondly, the KGFΔ33deletion variant removes a poly-serine stretch which is not present inKGF-1 and does not appear to be important for activity, but may hinderexpression of the protein in E. coli. Thus, removal of the poly-serinestretch may increase expression levels of active KGF-2 protein. Thirdly,expression of KGFΔ33 in E. coli, results in natural cleavage of KGF-2between residues 68 and 69. Thus, it is anticipated that KGF2 Δ33 willbe processed efficiently and will be stable in E. coli.

Construction of KGF2Δ33 in pQE6

To permit Polymerase Chain Reaction directed amplification andsub-cloning of KGF2Δ33 into the E. coli protein expression vector, pQE6,two oligonucleotide primers (5952 and 19138) complementary to thedesired region of KGF2 were synthesized with the following basesequence.

Primer 5952: 5′GCGGCACATGTCTTACAACCACCTGCAGGGTG 3′ (SEQ ID NO:91) Primer19138: 5′GGGCCCAAGCTTATGAGTGTACCACCAT 3′ (SEQ ID NO:92)

In the case of the N-terminal primer (5952), an AflIII restriction sitewas incorporated, while in the case of the C-terminal primer (19138) aHindIII restriction site was incorporated. Primer 5952 also contains anATG sequence adjacent and in frame with the KGF2 coding region to allowtranslation of the cloned fragment in E. coli, while primer 19138contains two stop codons (preferentially utilized in E. coli) adjacentand in frame with the KGF2 coding region which ensures correcttranslational termination in E. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature KGF-2 (aa 36–208) (constructed in Example 12C) as template.The resulting amplicon was restriction digested with AflIII and HindIIIand subcloned into NcoI/HindIII digested pQE6 protein expression vector.

Construction of KGF2Δ33 in pHE1

To permit Polymerase Chain Reaction directed amplification andsubcloning of KGF2Δ33 into the E. coli expression vector, pHE1, twooligonucleotide primers (6153 and 6150) corresponding to the desiredregion of KGF2 were synthesized with the following base sequence.

Primer 6153: 5′CCGGCGGATCCCATATGTCTTACAACCACCTGCAGG 3′ (SEQ ID NO:93)Primer 6150: 5′CCGGCGGTACCTTATTATGAGTGTACCACCATTGG 3′ (SEQ ID NO:94)

In the case of the N-terminal primer (6153), an NdeI restriction sitewas incorporated, while in the case of the C-terminal primer (6150) anAsp718 restriction site was incorporated. Primer 6153 also contains anATG sequence adjacent and in frame with the KGF2 coding region to allowtranslation of the cloned fragment in E. coli, while primer 6150contains two stop codons (preferentially utilized in E. coli) adjacentand in frame with the KGF2 coding region which ensures correcttranslational termination in E. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature KGF-2 (aa 36–208) (constructed in Example 12C) as template.The resulting amplicon was restriction digested with NdeI and Asp718 andsubcloned into NdeI/Asp718 digested pHE1 protein expression vector.

Nucleotide Sequence of KGF2Δ33:

ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTC (SEQ ID NO:95)TCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAA

Amino Acid Sequence of KGF A33:

MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEI (SEQ ID NO:96)GVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMVVHSB. Construction of an Optimized KGF-2 Δ33

In order to increase the expression levels of KGF2Δ33 in E. coli, thecodons of the complete gene were optimized to match those most highlyused in E. coli. As the template utilised to generate the KGF2Δ33 wascodon optimized within the N-terminal region, the C-terminal amino acids(84–208) required optimization.

Firstly, amino acids 172–208 were codon optimized to generateKGF2Δ33(s172–208). This was achieved by an overlapping PCR strategy.Oligonucleotides PM07 and PM08 (corresponding to amino acids 172–208)were combined and annealed together by heating them to 70° C. andallowing them to cool to 37° C. The annealed oligonucleotides were thenutilized as template for a standard PCR reaction which was directed byprimers PM09 and PM10. In a separate PCR reaction following standardconditions well known to those skilled in the art and using KGF2Δ33 astemplate, oligonucleotides PM05 (which overlaps with the PstI sitewithin coding region of KGF2) and PM11 were used to amplify the regionof KGF2 corresponding to amino acids 84–172. In a third PCR reaction,the product of the first PCR reaction (corresponding to codon optimizedamino acids 172–208) and the product of the second PCR reaction(corresponding to codon non-optimized amino acids 84–172) were combinedand used as template for a standard PCR reaction directed byoligonucleotides PM05 and PM10. The resulting amplicon was digested withPstI/HindIII and sub-cloned into PstI/HindIII digested pQE6KGF2Δ33,effectively substituting the corresponding non codon optimized region,and creating pQE6KGF2Δ33(s172–208).

To complete the codon optimization of KGF2, a synthetic gene codonoptimized for the region of KGF2 corresponding to amino acids 84–172 wasgenerated utilising overlapping oligonucleotides. Firstly, fouroligonucleotides (PM31, PM32, PM33 and PM 34) were combined and sevencycles of the following PCR was performed: 94° C., 30 secs; 46.5° C., 30secs; and 72° C., 30 secs.

A second PCR reaction directed by primers PM35 and PM36 was thenperformed following standard procedures, utilizing 1 μl of the first PCRreaction as template. The resulting codon optimized gene fragment wasthen digested with Pst1/Sal1 and subcloned into Pst1/Sal1 digestedpQE6KGF2Δ33(s172–208) to create a fully optimized KGF2 encoding gene,pQE6KGF2Δ33s.

To create an alternative E. coli protein expression vector, KGF2Δ33s wasPCR amplified utilising primers PM102 and PM130 on pQE6KGF2Δ33s. Theresulting amplicon was digested with NdeI and EcoRV and subcloned intothe pHE1 expression vector which had been digested with NdeI and Asp718(blunt ended) to create pHEΔ33s.

Oligonucleotide Sequences used in construction of codon optimizedKGF2Δ33s:

PM05: CAACCACCTGCAGGGTGACG (SEQ ID NO:97) PM07:AACGGTCGACAAATGTATGTGGCACTGAACGGTAAAGGTGCTCCAC (SEQ ID NO:98)GTCGTGGTCAGAAAACCCGTCGTAAAAACACC PM08:GGGCCCAAGCTTAAGAGTGTACCACCATTGGCAGAAAGTGAGCAG (SEQ ID NO:99)AGGTGTTTTTACGACGGGTTTTCTGACCACG PM09: GCCACATACATTTGTCGACCGTT (SEQ IDNO:100) PM10: GGGCCCAAGCTTAAGAGTG (SEQ ID NO:101) PM11:GCCACATACATTTGTCGACCGTT (SEQ ID NO:102) PM31:CTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCCTTCACCAAAT (SEQ ID NO:103)ACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGTACCAAG PM32:AGCTTTAACAGCAACAACACCGATTTCAACGGAGGTGATTTCCAGG (SEQ ID NO:104)ATGGAGTACGGGCAGTTTTCTTTCTTGGTACCAGAAACTTTACC PM33:GGTGTTGTTGCTGTTAAAGCTATCAACTCCAACTACTACCTGGCTAT (SEQ ID NO:105)GAACAAGAAAGGTAAACTGTACGGTTCCAAAGAATTTAACAAC PM34:GTCGACCGTTGTGCTGCCAGTTGAAGGAAGCGTAGGTGTTGTAACC (SEQ ID NO:106)GTTTTCTTCGATACGTTCTTTCAGTTTACAGTCGTTGTTAAATTCTTT GGAACC PM35:GCGGCGTCGACCGTTGTGCTGCCAG (SEQ ID NO:107) PM36:GCGGCCTGCAGGGTGACGTTCGTTGG (SEQ ID NO:108) PM102:CCGGCGGATCCCATATGTCTTACAACCACCTGCAGG (SEQ ID NO:109) PM130:CGCGCGATATCTTATTAAGAGTGTACCACCATTG (SEQ ID NO:110)

Nucleotide Sequence of KGF2Δ33(s172–208):

ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTC (SEQ ID NO:111)TCCTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGTACCAAGAAAGAAAACTGCCCGTACTCCATCCTGGAAATCACCTCCGTTGAAATCGGTGTTGTTGCTGTTAAAGCTATCAACTCCAACTACTACCTGGCTATGAACAAGAAAGGTAAACTGTACGGTTCCAAAGAATTTAACAACGACTGTAAACTGAAAGAACGTATCGAAGAAAACGGTTACAACACCTACGCTTCCTTCAACTGGCAGCACAACGGTCGACAAATGTATGTGGCACTGAACGGTAAAGGTGCTCCACGTCGTGGTCAGAAAACCCGTCGTAAAAACACCTCTGCTCACTTTCTGCCAATGGTGGTACACTCTTAA

Amino Acid Sequence of KGF2Δ33(s172–208):

MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEI (SEQ ID NO:112)GVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMVVHSC. Construction of N-Terminal Deletion Mutant KGF-2 Δ4

To increase the level of expression of KGF2 in E. coli and to enhancethe stability and solubility properties of E. coli expressed KGF2, adeletion variant KGF2Δ4 (amino acids 39–208) which removes the first 38amino acids of pre-processed KGF2 was constructed, including thecysteine at position 37. As the resulting KGF2 deletion moleculecontains an even number of cysteines, problems due to aggregation causedby intra-molecular disulphide bridge formation should be reduced,resulting in an enhanced level of expresssion of active protein.

To permit Polymerase Chain Reaction directed amplification andsub-cloning of KGF2Δ4 into the E. coli protein expression vector, pQE6,two oligonucleotide primers (PM61 and 19138) were synthesized with thefollowing base sequence.

PM61: CGCGGCCATGGCTCTGGGTCAGGACATG (SEQ ID NO:113) 19138:GGGCCCAAGCTTATGAGTGTACCACCAT (SEQ ID NO:114)

In the case of the N-terminal primer (PM61), an NcoI restriction sitewas incorporated, while in the case of the C-terminal primer (19138) aHindIII restriction site was incorporated. PM61 also contains an ATGsequence adjacent and in frame with the KGF2 coding region to allowtranslation of the cloned fragment in E. coli, while 19138 contains astop codon (preferentially utilized in E. coli) adjacent to and in framewith the KGF2 coding region which ensures correct translationaltermination in E. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the full length KGF2 (aa36–208) as template (constructed in Example 12C). The resulting ampliconwas restriction digested with NcoI and HindIII and subcloned intoNcoI/HindIII digested pQE6 protein expression vector.

Nucleotide Sequence of KGF2Δ4:

ATGGCTCTGGGTCAAGATATGGTTTCTCCGGAAGCTACCAACTCTTCC (SEQ ID NO:115)TCTTCCTCTTTCTCTTCCCCGTCTTCCGCTGGTCGTCACGTTCGTTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAA

Amino Acid Sequence of KGF2Δ4:

MALGQDMVSPEATNSSSSSFSSPSSAGRHVRSYNHLQGDVRWRKLFSFT (SEQ ID NO:116)KYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGA PRRGQKTRRKNTSAHFLPMVVHS

EXAMPLE 17 KGF-2 Δ33 Stimulated Wound Healing in Normal Rat

To demonstrate that KGF-2 Δ33 would accelerate the healing process,wound healing of excisional wounds were examined using the followingmodel.

A dorsal 6 mm excisional wound is created on Sprague Dawley rats (n=5)with a Keyes skin punch. The wounds are left open and treated topicallywith various concentrations of KGF-2 Δ33 (in 40 mM NaOAc and 150 mMNaCl, pH 6.5 buffer) and buffer (40 mM NaOAc and 150 mM NaCl, pH 6.5)for 4 days commencing on the day of wounding. Wounds are measured dailyusing a calibrated Jameson caliper. Wound size is expressed in squaremillimeters. On the final day wounds were measured and harvested forfurther analysis. Statistical analysis was done using an unpaired t test(mean±SE). Evaluation parameters include percent wound closure,histological score (1–3 minimal cell accumulation, no granulation; 4–6immature granulation, inflammatory cells, capillaries; 7–9 granulationtissue, cells, fibroblasts, new epithelium 10–12 mature dermis withfibroblasts, collagen, epithelium), re-epithelialization andimmunohistochemistry.

At three days postwounding, treatment with KGF-2 Δ33 displayed adecrease in wound size (30.4 mm² at 4 μg, p=0.006, 33.6 mm² at 1 μg,p=0.0007) when compared to the buffer control of 38.9 mm². At day fourpostwounding, treatment with KGF-2 Δ33 displayed a decrease in woundsize (27.2 mm² at 0.1 μg p=0.02, 27.9 mm² at 0.4 μg p=0.04) whencompared to buffer control of 33.8 mm². At day five postwounding,treatment with KGF-2 Δ33 displayed a decrease in wound size (18.1) mm²at 4 μg p=0.02 when compared to buffer control of 25.1 mm². See FIG. 36.

Following wound harvest on day 5, additional parameters were evaluated.KGF-2 Δ33 displayed an increase in the percentage of wound closure at 4μg (71.2%, p=0.02) when compared to buffer control 60.2%. Administrationof KGF-2 Δ33 also results in an improvement in histological score at 1and 4 μg (8.4 at 1 μg p=0.005, 8.5 at 4 μg p=0.04) relative to buffercontrol of 6.4. Re-epithelialization was also improved at 1 and 4 μgKGF-2 Δ33 (1389 μm at 1 μg p=0.007, 1220 μm at 4 μg p=0.02) relative tothe buffer control of 923 μm. See FIG. 37.

This study demonstrates that daily treatment with KGF-2 Δ33 acceleratesthe rate of wound healing in normal animals as shown by a decrease inthe gross wound area. In addition, the histological evaluation of woundsamples and assessment of re-epithelialization also show that KGF-2 Δ33improves the rate of healing in this normal rat model.

EXAMPLE 18 KGF-2 Δ33 Effect on Tensile Strength and Epidermal Thicknessin Normal Rat

To demonstrate that KGF-2 Δ33 would increase tensile strength andepidermal thickness of wounds the following experiment was performed.

A 2.5 cm full thickness midline incisional wound is created on the backof male Sprague Dawley rats (n=8 or 9). Skin incision is closed using 3equidistant metal skin staples. Buffer (40 mM NaOAc and 150 mM NaCl, pH6.5) or KGF-2 Δ33 (in 40 mM NaOAc and 150 mM NaCl, pH 6.5 buffer) weretopically applied at the time of wounding. Four wound strips measuring0.5 cm in width are excised at day 5. Specimens are used for the studyof breaking strength using an Instron™ skin tensiometer, hydroxyprolinedetermination and histopathological assessment. Breaking strength wasdefined as the greatest force withheld by each wound prior to rupture.Statistical analysis was done using an unpaired t test (mean±SE).

In an incisional skin rat model, topically applied KGF-2 Δ33 exhibited astatistically significant increase in breaking strength, tensilestrength and epidermal thickness as a result of a single intraincisionalapplication subsequent to wounding. In one study, the breaking strengthof KGF-2 treated wounds at 1,4, and 10 μg was significantly higher whencompared to the buffer controls (107.3 g at 1 μg p=0.0006, 126.4 g at 4μg p<0.0001, 123.8 g at 10 μg p<0.0001). See FIG. 38.

Epidermal thickness was assessed under light microscopy on MassonTrichrome sections. KGF-2 Δ33 treated wounds displayed increasedepidermal thickening (60.5μ at 1 μg, 66.51μ at 4 μg p=0.01, 59.6μ at 10μg) in contrast with the buffer control of 54.8μ. See FIG. 39.

These studies demonstrate that a single intraincisional application ofKGF-2 augments and accelerates the wound healing process characterizedby an increase in breaking strength and epidermal thickness ofincisional wounds.

EXAMPLE 19 KGF-2 Δ33 Effect on Normal Rat Skin

In order to determine the effect of KGF-2 Δ33 on normal rat skinfollowing intradermal injection the following experiment was performed.

Male adult SD rats (n=3) received six intradermal injections of eitherplacebo or KGF-2 Δ33 (in 40 mM NaOAc and 150 mM NaCl, pH 6.5 buffer) ina concentration of 1 and 4 μg in 50 μl on day 0. Animals were injectedwith 5-2′-bromo-deoxyrudine (BrdU) (100 mg/kg i.p.) two hours prior tosacrifice at 24 and 48 hours. Epidermal thickness was measured from thegranular layer to the bottom of the basal layer. Approximately, 20measurements were made along the injection site and the mean thicknessquantitated. Measurements were determined using a calibrated micrometeron Masson Trichrome stained sections under light microscopy. BrdUscoring was done by two blinded observers under light microscopy usingthe following scoring system: 0–3 none to minimal BrdU labeled cells;4–6 moderate labeling; 7–10 intense labeled cells. Animals weresacrificed 24 and 48 hours post injection. Statistical analysis was doneusing an unpaired t test. (mean±SE).

KGF-2 Δ33 treated skin displayed increased epidermal thickening at 24hours (32.2 μat 1 μg p<0.001, 35.4μ at 4 μg p<0.0001) in contrast withthe buffer control of 27.1μ. At 48 hours KGF-2 Δ33 treated skindisplayed increased epidermal thickening (34.0μ at 1 μg p=0.0003, 42.4μat 4 μg p<0.0001) compared to buffer control of 27.8μ. See FIG. 40.KGF-2 Δ33 treated skin also displayed increased BrdU immunostaining at48 hours (4.73 at 1 μg p=0.07, 6.85 at 4 μg p<0.0001) compared to buffercontrol of 3.33. See FIG. 41.

These studies demonstrate that a intradermal injection of KGF-2 augmentsand accelerates epidermal thickening. Thus, KGF-2 would haveapplications to prevent or alleviate wrinkles, improve aging skin andreduce scaring or improve healing from cosmetic surgery. In addition,KGF-2 can be used prophylactically to prevent or reduce oral mucosistis(mouth ulcers), intestinal inflammation in response to chemotherapy orother agents.

EXAMPLE 20 Anti-Inflammatory Effect of KGF-2 on PAF-Induced Paw Edema

To demonstrate an anti-inflammatory effect of KGF-2 the followingexperiment was performed using PAF-induced paw edema inflammation model.

Groups of four lewis rats (190˜210 gm) were injected subcutaneously inthe foot pad of the right hind paw with 120 μl solution containing 2.5nMol of PAF, together with the following reagents: 125 μg of Ckb-10(B5),24 μg of LPS, 73 μg of KGF-2 (Thr (36)—Ser (208) of FIGS. 1A–1C (SEQ IDNO:2) with a N-terminal Met) or no protein. The left hind paws weregiven the same amount of buffer to use as parallel control. Paw volumewas quantified immediately before, or 30 and 90 minutes after PAFinjection using a plethysmograph system. Percent (%) change of pawvolume were calculated.

Testing reagents in experiment No. 1 and No. 2 Groups PAF(R.) Ckβ-10(R.)LPS(R.) KGF-2(R.) (N = 4) 2.5 nMol 1.04 mg/ml 200 μg/ml 0.73 mg/mlBuffer 1 20 μl — — — 100 μl 2 20 μl 100 μl — — — 3 20 μl — 100 μl — — 420 μl — — 100 μl —

As shown in FIG. 42, right hind paws injected with PAF alone resulted ina significant increase in paw volume (75 or 100% for experiment No. 1 orNo. 2, respectively) at 0.5 hour post injection as expected; while lefthind paws receiving buffer or right hind paws receiving LPS or SEB aloneshow little sign of edema (data not shown). However, when KGF-2 wasgiven together with PAF locally, there is a substantial reduction (25 or50% for experiment No. 1 or No. 2, respectively) in paw volume comparedwith PAF alone-challenged paws. The reduction of paw edema was notobserved in animal receiving PAF together with Ckb-10 (a differentprotein), LPS or SEB (two inflammatory mediators). These results suggestthat the anti-inflammatory effect of KGF-2 is specific and not due tosome non-specific nature of the protein.

Effect of KGF-2 Δ33 on PAF-Induced Paw Edema in Rats

Following the experiments described above with KGF-2 Δ33 to confirm itsin vitro biological activities for stimulating keratinocyteproliferation and its in vivo effect on wound healing, KGF-2 Δ33 wasfurther evaluated in the PAF-induced paw edema model in rats. Groups offour Lewis rats (190˜210gm) were injected subcutaneously in the foot padof the right hind paw with 120 μl solution containing 2.5 nMol of PAF,together with 210 μg of KGF-2 Δ33 or albumin. The left hind paws weregiven the same amount of buffer, albumin or KGF-2 Δ33 alone to use asparallel control. Paw volume was quantified at different intervals afterPAF injection using a plethysmograph system. Percent (%) change of pawvolume was calculated.

As shown in FIG. 43, right hind paws injected with PAF and albuminresulted in a significant increase (75%) in paw volume at 0.5 hour postinjection as expected; while left hind paws receiving buffer, albumin orKGF-2 Δ33 alone showed little sign of edema. However, when KGF-2 Δ33 wasgiven together with PAF locally, there was a substantial reduction(average 20%) in paw volume, when compared with PAF plusalbumin-challenged paws, throughout the entire experiment which wasended in 4 hours. These results confirm the anti-tory inflammatoryproperty of KGF-2 Δ33.

Testing Reagents Groups PAF Albumin KGF-2 Δ33 (N = 4) 2.5 nMol 2.1 mg/ml2.1 mg/ml Buffer 1 20 μl 100 μl — — 2 20 μl — 100 μl — 3 — 120 μl — — 4— — 120 μl — 5 — — — 120 μl

Thus, KGF-2 is useful for treating acute and chronic conditions in whichinflammation is a key pathogenesis of the diseases including but notlimiting to psoriasis, eczema, dermatitis and/or arthritis.

EXAMPLE 21 Effect of KGF-2 Δ33 on End-to-End Colonic Anastomosis RatModel

This example demonstrates that KGF-2 Δ33 will increase the rate ofintestinal repair in a model of intestinal or colonic anastomosis inWistar or Sprague Dawley rats. The use of the rat in experimentalanastomosis is a well characterized, relevant and reproducible model ofsurgical wound healing. This model can also be extended to study theeffects of chronic steriod treatment or the effects of variouschemotherapeutic regimens on the quality and rate of surgical woundhealing in the colon and small intestine (Mastboom W. J. B. et al. Br.J. Surg. 78: 54–56 (1991), Salm R. et al. J. Surg. Oncol. 47: 5–11,(1991), Weiber S. et al. Eur. Surg. Res. 26: 173–178 (1994)). Healing ofanastomosis is similar to that of wound healing elsewhere in the body.The early phases of healing are characterized by acute inflammationfollowed by fibroblast proliferation and synthesis of collagen. Collagenis gradually modeled and the wound is strengthened as new collagen issynthesized. (Koruda M. J., and Rolandelli, R. H. J. Surg. Res. 48:504–515 (1990). Most postoperative complications such as anastomoticleakage occur during the first few days following surgery—a periodduring which strength of the colon is mainly secured by the ability ofthe wound margin to hold sutures. The suture holding capacity of the GItract has been reported to decrease by as much as 80% during the firstpostoperative days (Hogstrom H and Haglund U. Acta Chir Scand 151:533–535 (1985), Jonsson K, et al. Am J. Surg. 145: 800–803 (1983)).

Male adult SD rats (n=5) were anesthetized with a combination ofketamine (50 mg/kg) and xylazine (5 mg/kg) intramuscularly. Theabdominal cavity was opened with a 4 cm long midline incision. A 1 cmwide segment of the left colon was resected 3 cm proximal to theperitoneal reflection while preserving the marginal vessels. A singlelayer end-to-end anastomosis was performed with 8-10 interrupted 5-0Vicryl inverted sutures to restore intestinal continuity. Theanastomosis was then topically treated via syringe with either buffer orKGF-2 Δ33 at concentrations of 1 and 4 μg. The incisional wound wasclosed with 3-0 running silk suture for the muscle layer and surgicalstaples for the skin. Treatments were then administered daily thereafterand consisted of buffer or KGF-2 Δ33 and 1 and 5 mg/kg sc. Weights weretaken on the day of surgery and daily thereafter. Animals wereeuthanized 24 hours following the last treatment (day 5). Animals wereanesthetized and received barium enemas and were x-rayed at a fixeddistance. Radiologic analysis following intracolonic administration by 2blinded observers revealed that KGF-2 Δ33 treated groups had 1) adecreased rate of barium leakage at the surgical site, 2) lesser degreeof constriction at the surgical site, and 3) an increase in the presenceof fecal pellets distal to the surgical site.

Colonic Anastomosis Radiologic Analysis Feces Anastomotic ProximalPeritoneal Groups Present Constriction Distension Leakage No Treatment20% 80% 80% 60% (N = 5) Buffer 40% 60% 80% 75% (N = 5) KGF-2 Δ33 60% 20%100%  20% [1 mg/kg] (N = 5) KGF-2 Δ33 100%   0% 75% 25% [5 mg/kg] (N =4)

EXAMPLE 22 Construction of Carboxy Terminal Mutations in KGF-2

The carboxyl terminus of KGF-2 is highly charged. The density of thesecharged residues may affect the stability and consequently thesolubility of the protein. To produce muteins that might stabilize theprotein in solution a series of mutations were created in this region ofthe gene.

To create point mutants 194 R/E, 194 R/Q, 191 K/E, 191 K/Q, 188R/E,188R/Q, the 5952 KGFΔ33 5′ AflIII 5′ primer was used with the indicated3′ primers, which contain the appropriate point mutations for KGF-2, inPCR reactions using standard conditions well known to those skilled inthe art with KGF-2 Δ33 as template. The resulting products wererestricted with AflIII and Hind III and cloned into the E. coliexpression vector, pQE60 restricted with NcoI and Hind III.

5952 KGF Δ 33 5′ Afl III: 5′ GCGGCACATGTCTTACAACCACCTGCAGGGTG 3′ (SEQ IDNO:117) KGF2 3′HindIII 194aa R to E:5′CTGCCCAAGCTTTTATGAGTGTACCACCATTGGAAGAAAGTGAGC (SEQ ID NO:118)AGAGGTGTTTTTTTCTCGTGTTTTCTGTCC 3′

KGF2Δ33,194 R/E Nucleotide Sequence:

ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTC (SEQ ID NO:119)TCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAGAAAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG

KGF2Δ33,194 R/E Amino Acid Sequence:

MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEI (SEQ ID NO:120)GVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQKTR E KNTSAHFLPMVVHS

KGF2Δ33,194 R/Q Construction:

The following primers were used:

5952 KGF Δ33 5′ Af1 III: 5′ GCGGCACATGTCTTACAACCACCTGCAGGGTG 3′ (SEQ IDNO:121) KGF2 3′ HindIII 194 aa R to Q5′CTGCCCAAGCTTTTATGAGTGTACCACCATTGGAAGAAAGTGAGCAG (SEQ ID NO:122)AGGTGTTTTTCTGTCGTGTTTTCTGTCC 3′

KGF2Δ33,194 R/Q Nucleotide Sequence:

ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTC (SEQ ID NO:123)TCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGACAGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG

KGF2Δ33,194 R/Q Amino Acid Sequence:

MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEI (SEQ ID NO:124)GVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQKTR Q KNTSAHFLPMVVHS

KGF2Δ33,191 K/E Construction:

The following primers were used:

5952 KGF Δ 33 5′ Af1 III: 5′ GCGGCACATGTCTTACAACCACCTGCAGGGTG 3′ (SEQ IDNO:125) KGF2 3′ HindIII 191aa K to E5′CTGCCCAAGCTTTTATGAGTGTACCACCATTGGAAGAAAGTGAGCAG (SEQ ID NO:126)AGGTGTTTTTCCTTCGTGTTTCCTGTCCTCTCCTTGG 3′

KGF2Δ33,191 K/E Nucleotide Sequence:

ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTC (SEQ ID NO:127)TCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGGAAACACGAA GGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG

KGF2Δ33,191 K/E Amino Acid Sequence:

MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEI (SEQ ID NO:128)GVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQ E TRRKNTSAHFLPMVVHS

KGF2Δ33, 191 K/Q Construction:

The following primers were used:

5952 KGFΔ33 5′ Af1 III: 5′GCGGCACATGTCTTACAACCACCTGCAGGGTG 3′ (SEQ IDNO:129) KGF2 3′ HindIII 191aa K to Q5′CTGCCCAAGCTTTTATGAGTGTACCACCATTGGAAGAAAGTGAGCAG (SEQ ID NO:130)AGGTGTTTTTCCTTCGTGTCTGCTGTCCTCTCCTTGG 3′

KGF23′ HindIII 191 K/Q Nucleotide Sequence:

ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTC (SEQ ID NO:131)TCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGCAGACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG

KGF2Δ33, 191 K/Q Amino Acid Sequence:

MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEI (SEQ ID NO:132)GVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQ Q TRRKNTSAHFLPMVVHS

KGF2Δ33, 188R/E Construction:

The following primers were used:

5952 KGFΔ33 5′ Af1 III: 5′ GCGGCACATGTCTTACAACCACCTGCA (SEQ ID NO:133)GGGTG 3′ KGF2 3′ HindIII 188aa R to E: 5′CTGCCCAAGCTTTTATGAGTGTACCAC(SEQ ID NO:134) CATTGGAAGAAAGTGAGCAGAGGTGTTTTTCCTTCGTGTTTTCTGTCCTTCCCTTGGAGC TCCTTT 3′

KGF2Δ33, 188R/E Nucleotide Sequence:

ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTC (SEQ ID NO:135)TCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGGAAGGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG

KGF2Δ33, 188R/E Amino Acid Sequence:

MYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIG (SEQ ID NO:136)VVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFN WQHNGRQMYVALNGKGAPR EGQKTRRKNTSAHFLPMVVHS

KGF2Δ33, 188 R/Q Construction:

The following primers were used:

5952 KGF Δ33 5′ Afl III: 5′ GCGGCACATGTCTTACAACCACCTGCAGGGTG 3′ (SEQ IDNO:137) KGF2 3′ HindIII 188aa R to Q:5′CTGCCCAAGCTTTTATGAGTGTACCACCATTGGAAGAAAGTGAGCAG (SEQ ID NO:138)AGGTGTTTTTCCTTCGTGTTTTCTGTCCCTGCCTTGGAGCTCCTTT 3′

KGF2Δ33, 188 R/Q Nucleotide Sequence:

ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTC (SEQ ID NO:139)TCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGCAGGGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG

KGF2Δ33, 188 R/Q Amino Acid Sequence:

MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEI (SEQ ID NO:140)GVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASF NWQHNGRQMYVALNGKGAPR QGQKTRRKNTSAHFLPMVVHS

KGF2Δ33, 183K/E Construction:

For mutation 183K/E, two PCR reactions were set up for oligonucleotidesite directed mutagenesis of this lysine. In one reaction, 5952 KGFΔ335′ AflIII was used as the 5′ primer, and KGF2 183aa K to E antisense wasused as the 3′ primer in the reaction. In a second reaction, KGF2 5′183aa K to E sense was used as the 5′ primer, and KGF2 3′ HindIII TAAstop was used as the 3′ primer. KGF-2 Δ33 was used as template for thesereactions. The reactions were amplified under standard conditions wellknown to those skilled in the art. One microliter from each of these PCRreactions was used as template in a subsequent reaction using, as a 5′primer, 5453 BspHI, and as a 3′ primer, 5258 HindIII. Amplification wasperformed using standard conditions well known to those skilled in theart. The resulting product was restricted with AflIII and HindIII andcloned into the E. coli expression vector pQE60, which was restrictedwith NcoI and HindIII.

The following primers were used:

5952 KGF Δ33 5′ Afl III: (SEQ ID NO:141) 5′GCGGCACATGTCTTACAACCACCTGCAGGGTG 3′ KGF2 5′ 183aa K to E sense: (SEQ IDNO:142) 5′ TTGAATGGAGAAGGAGCTCCA 3′ KGF2 183aa K to E antisense: (SEQ IDNO:143) 5′ TGGAGCTCCTTCTCCATTCAA 3′ KGF2 3′ HindIII TAA stop: (SEQ IDNO:144) 5′ CTGCCCAAGCTT TTATGAGTGTACCACCATTGG 3′

KGF2Δ33, 183K/E Nucleotide Sequence:

ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTC (SEQ ID NO:145)TCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAGAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG

KGF2Δ33, 183K/E Amino Acid Sequence:

MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEI (SEQ ID NO:146)GVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASF NWQHNGRQMYVALNG EGAPRRGQKTRRKNTSAHFLPMVVHS

EXAMPLE 23 Effect of KGF-2 on Survival After Total Body Irradiation inBalb/c Mice

Ionizing radiation is commonly used to treat many malignancies,including lung and breast cancer, lymphomas and pelvic tumors (Ward, W.F. et al., CRC Handbook of Animal Models of Pulmonary Disease, CRCPress, pp. 165–195 (1989)). However, radiation-induced injury (lung,intestine, etc.) limits the intensity and the success of radiationtherapy (Morgan, G. W. et al., Int. J. Radiat. Oncol. Biol. Phys. 31:361(1995)). The gastrointestinal mucosa has a rapid cell cycle and isparticularly sensitive to cytotoxic agents (Potten, C. S., et al., In:Cytotoxic Insult to Tissue, Churchill Livingstone, pp. 105–152 (1983)).Some of the manifestations of intestinal radiation damage include acuteproctitis, intestinal fibrosis, stricture or fistula formation(Anseline, D. F. et al. Ann. Surg. 194:716–724 (1981)). A treatmentwhich protects normal structures from radiation without altering theradiosensisitivity of the tumor would be beneficial in the management ofthese disorders. Regardless of the irradiated area, the dose ofradiation is limited by the radiosensitivity of normal tissue.Complications following total or partial body irradiation includepneumonitis, fibrosis, gastro-intestinal injury and bone marrowdisorders.

Several cytokines including IL-1, TNF, IL-6, IL-12 have demonstratedradioprotective effects following TBI (Neta, R. et al., J. Exp. Med.173:1177 (1991)). IL-11 has been shown to protect small intestinalmucosal cells after combined irradiation and chemotherapy (Du, X. X. etal., Blood 83:33 (1994)) and radiation-induced thoracic injury (Redlich,C. A. et al. The Journal of Immunology 157:1705–1710 (1996)).

Animals

All experiments were performed using BALB/c mice. Animals were purchasedat 6 weeks of age and were 7 weeks old at the beginning of the study.All manipulations were performed using aseptic techniques. This studywas conducted according to the guidelines set forth by the Human GenomeSciences, Inc., Institutional Animal Care and Use Committee whichreviewed and approved the experimental protocol.

KGF-2

The protein consists of a 141 amino acid human protein termed KGF-2 Δ33.This protein is a truncated isoform of KGF-2 that lacks the first 33amino-terminal residues of the mature protein. The gene encoding thisprotein has been cloned into an E. coli expression vector. Fractionscontaining greater that 95% pure recombinant materials were used for theexperiment. KGF-2 was formulated in a vehicle containing 40 mM NaAcetate+150 mM NaCl, pH 6.5. Dilutions were made from the stock solutionusing the same vehicle.

Total Body Irradiation and Experimental Design

Mice were irradiated with 519 RADS (5.19 Gy) using a 68 Mark I ShepherdCesium Irradiator. The KGF-2 Δ33 was administered daily subcutaneously,starting 2 days before irradiation and continuing for 7 days afterirradiation. Daily weights were obtained in all mice. Groups of micewere randomized to receive one of three treatments: Total bodyirradiation (TBI) plus buffer, TBI plus KGF-2 Δ33 (1 mg/kg sq), TBI plusKGF-2 Δ33 (5 mg/kg sq). Two independent experiments were performed.

Results

Two studies were performed using irradiated animals. In the first study,animals were irradiated with 519 RADS (5.19 Gy). Animals were treatedwith buffer or KGF-2 Δ33 at 1 & 5 mg/kg, s.q. two days prior toirradiation and daily thereafter for 7 days. At day 25 after total bodyirradiation 1/5 animals survived in the buffer group. In contrast, KGF-2treated groups had 5/5 animals @ 1 mg/kg and 4/5 @ 5 mg/kg (FIG. 44).

In addition, KGF-2 treated animals displayed 0.9% and 5.3% weight gainat day 20 post-TBI. In contrast, the buffer treated group had 4.2%weight loss at day 20. Normal non-irradiated age matched control animalsshowed 6.7% weight gain in the same time period (FIG. 45).

Animals in the second study were also irradiated with 519 RADS (5.19Gy). These animals were treated with buffer or KGF-2 Δ33 at 1 & 5 mg/kg,s.q. two days prior to irradiation and daily thereafter for 7 days. Atday 15 after total body irradiation all the animals in the buffer groupwere dead. KGF-2 at 1 mg/kg had 30% survival and 60% survival at 5mg/kg. At day 25 after TBI the 1 mg/kg group showed 20% survival and the5 mg/kg 50% survival (FIG. 46).

Conclusions

In summary, these results demonstrate that KGF-2 has a protective effectafter TBI. The ability of KGF-2 to increase survival rate of animalssubjected to TBI suggests that it would also be useful inradiation-induced injuries and to increase the therapeutic ratio ofirradiation in the treatment of malignancies.

EXAMPLE 24 Evaluation of KGF-2 in the TPA Model of CutaneousInflammation in Mice

To demonstrate that KGF-2 would attenuate the progression of contactdermatitis, a tetradecanoylphorbol acetate (TPA)-induced cutaneousinflammation model in mice is used. The use of the female BALB/c andmale Swiss Webster mice in experimental cutaneous inflammation arewell-characterized, relevant and reproducible models of contactdermatitis. These strains of mice have been shown to develop along-lasting inflammatory response, following topical application ofTPA, which is comprised of local hemodynamics, vascular permeability andlocal migration of leukocytes, and these pathological changes aresimilar to those of human dermatitis (Rao et al. 1993, Inflammation17(6):723; Rao et al. 1994, J. Kipid Mediators Cell Signalling 10:213).

Groups of mice receive either vehicle or KGF-2 intraperitoneally,sub-cutaneously, or intravenously 60 min after the topical applicationof TPA (4 μg/ear) applied as a solution in acetone (200 μg/ml), 10 μleach to the inner and outer surface of ear. The control group receives20 μl of acetone as a topical application. Four hours following theapplication of TPA, increase in ear thickness is measured and ears areexcised for histology. To determine vascular permeability in response toTPA, mice are intravenously injected through tail veins with Evans blue(300 mg/kg) at selected times after topical application of TPA and miceare sacrificed 15 min thereafter. Ears are excised and removed, thenextracted into dimethylformamide and centrifuged. Absorbance readingsare spectrophotometrically measured at 590 nm.

EXAMPLE 25 Effect of KGF-2 Δ33 in Wound Healing

The biological effects of KGF-2 Δ33 in the skin were examined based onthe initial in vitro data demonstrating KGF-2's capacity to stimulateprimary human epidermal keratinocytes as well as murine pro-B BaF3 cellstransfected with the FGFR isoform 2iiib. Initial experiments wereperformed to determine the biological effects of KGF-2 Δ33 followingintradermal administration. Following the intradermal studies, KGF-2 Δ33was explored in a variety of wound healing models (including fullthickness punch biopsy wounds and incisional wounds) to determine itspotential as a wound healing agent.

Effect of KGF-2 Δ33 in a Glucocorticoid-Impaired Rat Model of WoundHealing

Impaired wound healing is an important clinical problem associated witha variety of pathologic conditions such as diabetes and is acomplication of the systemic administration of steroids orantimetabolites. Treatment with systemic glucocorticoids is known toimpair wound healing in humans and in animal models of tissue repair. Adecrease in circulating monocyte levels and an inhibition of procollagensynthesis have been observed subsequent to glucocorticoidadministration. The inflammatory phase of healing and matrix synthesisare therefore important factors involved in the complex process oftissue repair. In the present study the effects of multiple topicalapplications of KGF-2 were assessed on full thickness excisional skinwounds in rats in which healing has been impaired by the systemicadministration of methylprednisolone.

Sprague Dawley rats (n=5/treatment group) received 8 mm dorsal woundsand methylprednisolone (17 mg/kg, i.m.) to impair healing. Wounds weretreated topically each day with buffer or KGF-2 at doses of 0.1, 0.5 and1.5 μg in a volume of 50 μl. Wounds were measured on days 2, 4, 6, and 8using a calibrated Jameson caliper. On day 6 (data not shown), and day 8(FIG. 47) KGF-2 treated groups showed a statistically significantreduction in wound closure when compared to the buffer control.

Effect of KGF-2 Δ33 on Wound Healing in a Diabetic Mouse Model

Genetically diabetic homozygous female (db+/db+) mice, 6 weeks of age(n=6), weighing 30–35 g were given a dorsal full thickness wound with a6 mm biopsy punch. The wounds were left open and treated daily withplacebo or KGF-2 at 0.1, 0.5 and 1.5 μg. Wound closure was determinedusing a Jameson caliper. Animals were euthanized at day 10 and thewounds were harvested for histology.

KGF-2 displayed a significantly improvement in percent wound closure at0.1 μg (p=0.02) when compared to placebo or with the untreated group.Administration of KGF-2 also resulted in an improvement in histologicalscore at 0.1 μg (p=0.03) when compared to placebo or with the untreatedgroup (p=0.01) and 1.5 μg (p=0.05) compared to the untreated group.

Conclusions

Based on the results presented above, KGF-2 shows significant activityin impaired conditions such as glucocorticoid administration anddiabetes. Therefore, KGF-2 may be clinically useful in stimulatinghealing of wounds after surgery, chronic ulcers in patients withdiabetes or poor circulation (e.g., venous insufficiency and venousulcers), burns and other abnormal wound healing conditions such asuremia, malnutrition, vitamin deficiencies and systemic treatment withsteroids and antineoplastic drugs.

EXAMPLE 26 Effects of KGF-2 Δ33 on Oral Mucosa

Cytotoxic agents used clinically have the unfortunate effect ofinhibiting the proliferation of the normal epithelia in some locations,such as the oral mucosa, leading to life-threatening disturbances in themucosal barrier. We have conducted studies to examine the efficacy ofKGF-2 in this clinical area. The data supports a therapeutic effect ofKGF-2 in models of mucositis.

Effects of KGF-2 Δ33 on Hamster Oral Mucosa

We sought to determine if KGF-2 might induce proliferation of normaloral mucosal epithelium. The effect of KGF-2 in the oral mucosa wasassessed in male Golden Syrian hamsters. The cheek pouch of the hamsterwas treated daily with buffer or KGF-2 Δ33 (at 0.1, 1 and 10 μg/cheek)which were applied topically to anesthetized hamster cheeks in a volumeof 100 μl per cheek. The compound was in contact with the cheek for aminimum of 60 seconds and subsequently swallowed. After 7 days oftreatment, animals were injected with BrdU and sacrificed as describedabove. Proliferating cells were labeled using anti-BrdU antibody. FIG.48 shows that there was a significant increase in BrdU labeling (cellproliferation) when animals were treated with 1 μg and 10 μg of KGF-2Δ33 (when compared to buffer treatment).

Topical treatment with KGF-2 induced the proliferation of normal mucosalepithelial cells. Based upon these results, KGF-2 may be clinicallyuseful in the prevention of oral mucositis caused by anychemotherapeutic agents (or other toxic drug regimens), radiationtherapy, or any combined chemotherapeutic-radiation therapy regimen. Inaddition, KGF-2 may be useful as a therapeutic agent by decreasing theseverity of damage to the oral mucosa as a result of toxic agents(chemotherapy) or radiotherapy.

EXAMPLE 27 The Effect of KGF-2 Δ33 on Ischemic Wound Healing in Rats

The aim of the experiments presented in this example was to determinethe efficacy of KGF-2 in wound healing using an ischemic wound healingmodel.

The blood supply of local skin was partially interrupted by raising of asingle pedicle full-thickness random myocutaneous flap (3×4 cm). Afull-thickness wound was made into the local skin, which is composed ofthe myocutaneous flap. Sixty, adult Sprague-Dawley rats were used andrandomly divided into treatments of KGF-2 Δ33 and placebo groups forthis study (5 animals/group/time-point). The wounds were harvestedrespectively at day 1, 3, 5, 7, 10 and 15 post-wounding.

The wound breaking strength did not show a significant differencebetween KGF-2 and buffer treated groups at early time points until day10 and 15 post-wounding.

The results indicated that KGF-2 improved significantly the woundbreaking strength in ischemic wound repair after 10 days post-wounding.These results also suggest that ischemia delays the healing process inboth groups compared to the data previously obtained in studies ofnormal wound healing.

This myocutaneous flap model supplies data and information in anischemic situation which results from venous return. These resultssuggest that KGF-2 could be used in the treatment of chronic venous legulcers caused by an impairment of venous return and/or insufficiency.

EXAMPLE 28 Evaluation of KGF-2 in the Healing of Colonic Anastomosis inRats

The results of the present experiment demonstrate that KGF-2 Δ33increases the rate of intestinal repair in a model of intestinal orcolonic anastomosis in Wistar or Sprague Dawley rats. In addition, thismodel can be used to demonstrate that KGF-2 and its isoforms increasethe capability of the gastrointestinal or colon wall to bind sutures.

The use of the rat in experimental anastomosis is a well characterized,relevant and reproducible model of surgical wound healing. This modelcan also be extended to study the effects of chronic steroid treatmentor the effects of various chemotherapeutic regimens on the quality andrate of surgical wound healing in the colon and small intestine(Mastboom, W. J. B. et al., Br. J. Surg. 78:54–56 (1991); Salm, R. etal., J. Surg. Oncol. 47:5–11 (1991); Weiber, S. et al., Eur. Surg. Res.26:173–178 (1994)). Healing of anastomosis is similar to that of woundhealing elsewhere in the body. The early phases of healing arecharacterized by acute inflammation followed by fibroblast proliferationand synthesis of collagen. Collagen is gradually modeled and the woundis strengthened as new collagen is synthesized (Koruda, M. J., andRolandelli, R. H., J. Surg. Res. 48:504–515 (1990)). Most postoperativecomplications such as anastomotic leakage occur during the first fewdays following surgery—a period during which strength of the colon ismainly secured by the ability of the wound margin to hold sutures. Thesuture holding capacity of the GI tract has been reported to decrease byas much as 80% during the first postoperative days (Hogstrom, H. andHaglund, U., Acta Chir. Scand. 151:533–535 (1985); Jonsson, K. et al.,Am J. Surg. 145:800–803 (1983)).

Rats were anesthetized with a combination of ketamine (50 mg/kg) andxylazine (5 mg/kg) intramuscularly. Animals were kept on a heating padduring skin disinfection, surgery, and post-surgery. The abdominalcavity was opened with a 4 cm long midline incision. A 1 cm wide segmentof the left colon was resected 3 cm proximal to the peritonealreflection while preserving the marginal blood vessels. A single layerend-to-end anastomosis was performed with 8-10 interrupted 8-0 propyleneinverted sutures which were used to restore intestinal continuity. Theincisional wound was closed with 3-O running silk suture for the musclelayer and surgical staples for the skin. Daily clinical evaluations wereconducted on each animal consisting of individual body weight, bodytemperature, and food consumption patterns.

KGF-2 Δ33 and placebo treatment were daily administered sc, topically,ip, im, intragastrically, or intracolonically immediately followingsurgery and were continued thereafter until the day of sacrifice, day 7.There was an untreated control, a placebo group, and KGF-2 Δ33 groups.Two hours prior to euthanasia, animals were injected with 100 mg/kg BrdUi.p. Animals were euthanized 24 hours following the last treatment (day5). A midline incision was made on the anterior abdominal wall and a 1cm long colon segment, including the anastomosis, was removed. A thirdsegment at the surgical site was taken for total collagen analysis.

In a series of two experiments, male adult SD rats (n=5) wereanaesthetized and received a single layer end-to-end anastomosis of thedistal colon with 8-10 interrupted 6-0 prolene inverted sutures. Theanastomotic site was then topically treated via syringe with eitherbuffer or KGF-2 Δ33 at concentrations of 1 and 4 μg. Animals were thentreated daily thereafter with either buffer or KGF-2 Δ33 atconcentrations of 1 mg/kg or 5 mg/kg ip. Animals were euthanized on day5 and the colon excised and snap frozen in liquid nitrogen, lyophilizedand subjected to collagen determinations. Collagen concentration isexpressed as μg collagen/mg dry weight tissue. Statistical analysis wasdone using an unpaired t test. Mean±SE. On day 5 rats were anesthetizedand subjected to barium enemas followed by radiographic analysis. Bariumenema radiologic assessment of end-to-end left colonic anastomosis fromtwo experiments showed a consistent reduction in peritoneal leakage withKGF-2 treated animals at 1 and 5 mg/kg. This data is shown in the Tablebelow. In addition, breaking strength at the site of surgery was alsoexamined using a tensiometer. No significant differences were observedbetween the KGF-2 Δ33 and buffer groups. As shown in FIG. 49,significant increases in collagen content at the surgical site weredemonstrated at both 1 mg/kg KGF-2 Δ33 (p=0.02) and 5 mg/kg (p=0.004)relative to buffer controls.

TABLE Colonic Anastomosis Radiologic Analysis Feces AnastomoticPeritoneal Groups Present Constriction* Leakage No Treatment 50% 2.0 75%(N = 8) Buffer 57% 1.0 50% (N = 7) KGF-2Δ33 50% 1.3 37% [1 mg/kg] (N =8) KGF-2Δ33 77% 1.6 11% [5 mg/kg] (N = 9) *Anastomotic ConstrictionScoring: 0 -no constriction; 1–5 -minimal to severe constriction

Male adult SD rats (n=5) were anesthetized with a combination ofketamine (50 mg/kg) and xylazine (5 mg/kg) intramuscularly. Theabdominal cavity was opened with a 4 cm long midline incision. A 1 cmwide segment of the left colon was resected 3 cm proximal to theperitoneal reflection while preserving the marginal vessels. A singlelayer end-to-end anastomosis was performed with 8-10 interrupted 6-0prolene inverted sutures to restore intestinal continuity. Theanastomosis was then topically treated via syringe with either buffer orKGF-2 at concentrations of 1 and 4%1 g. The incisional wound was closedwith 3-O running silk suture for the muscle layer and surgical staplesfor the skin. Treatments were then administered daily thereafter andconsisted of buffer or KGF-2 Δ33 at 1 and 5 mg/kg sc. Weights were takenon the day of surgery and daily thereafter. Animals were euthanized 24hours following the last treatment (day 5). Animals were anesthetizedand received barium enemas and were x-rayed at a fixed distance. Theanastomosis was then excised for histopathological and biomechanicalanalysis.

EXAMPLE 29 Evaluation of KGF-2 in a Model of Inflammatory Bowel Disease

KGF-2 is a protein that induces keratinocyte proliferation in vitro andis active in a variety of wound healing models in vivo. The purpose ofthis study was to determine whether KGF-2 was efficacious in a model ofmurine colitis induced by ad libitum exposure to dextran sodium sulfatein the drinking water.

Six to eight week old female Swiss Webster mice (20–25g, Charles River,Raleigh, N.C.) were used in a model of inflammatory bowel diseaseinduced with a 4% solution of sodium sulfate (DSS, 36,000–44,000 MW,American International Chemistry, Natick, Mass.)) administered adlibitum for one week. KGF-2 was given by daily parenteral administration(n=10). Three parameters were used to determine efficacy: 1) clinicalscore, based on evaluation of the stool; 2) histological score, based onevaluation of the colon; and 3) weight change. The clinical score wascomprised of two parts totaling a maximum of score of four. Stoolconsistency was graded as: 0=firm; 1=loose; 2=diarrhea. Blood in thestool was also evaluated on a 0 to 2 scale with 0=no blood; 1=occultblood; and 2=gross rectal bleeding. A mean group score above 3 indicatedprobable lethality, and disease which had progressed beyond itstreatable stage. Clinical scores were taken on Day 0, 4, 5, 6, and 7. Toarrive at a histological score, slides of the ascending, transverse anddescending colon were evaluated in a blinded fashion based oninflammation score (0–3) and crypt score (0–4). Body weight was measureddaily. Data was expressed as mean+SEM. An unpaired Student's t test wasused to determine significant differences compared to the diseasecontrol (* p<0.05; ** p<0.01; *** p<0.001).

When DSS-treated mice were given a daily, intra-peritoneal (IP)injection of KGF-2 Δ33 at a dose of 1, 5 or 10 mg/kg for 7 days, KGF-2significantly reduced clinical score, 28, 38 and 50 percent,respectively. Histological evaluation closely paralleled the dosedependent inhibition of the clinical score, with the 1, 5 and 10 mg/kgdose reducing histological score a significant 26, 48 and 51 percent.KGF-2 also significantly reduced weight loss associated with DSS-inducedcolitis.

In a second study, a comparison was made of the relative efficacy ofKGF-2 Δ33 (10 mg/kg) when given IP or sub-cutaneous (SC) daily. By theend of the experiment on Day 7, animals injected IP with KGF-2 had asignificant, 34 percent reduction in clinical score while KGF-2 injectedSC resulted in a significant 46 percent reduction. The SC dose alsosignificantly reduced weight loss over DSS controls. Based onmeasurement of clinical score and body weight, SC administration ofKGF-2 is at least as efficacious as IP administration.

EXAMPLE 30 Effects of KGF-2 Δ33 on Normal Urinary Bladder and Prostateand in Cyclophosphamide-Induced Hemorrhagic Cystitis in Rats

The purpose of this example is to show that KGF-2 Δ33 is capable ofstimulating urinary bladder proliferation in normal rats and that thereis a therapeutic effect of KGF-2 Δ33 in a rat model ofcyclophosphamide-induced hemorrhagic cystitis.

Some cytotoxic agents used clinically have side effects resulting in theinhibition of the proliferation of the normal epithelium in the bladder,leading to potentially life-threatening ulceration and breakdown in theepithelial lining of the bladder. For example, cyclophosphamide causeshemorrhagic cystitis in some patients, a complication which can besevere and in some cases fatal. Fibrosis of the urinary bladder may alsodevelop with or without cystitis. This injury is thought to be caused bycyclophosphamide metabolites excreted in the urine. Hematuria caused bycyclophosphamide usually is present for several days, but may persist.In severe cases medical or surgical treatment is required. Instances ofsevere hemorrhagic cystitis result in discontinued cyclophosphamidetherapy. In addition, urinary bladder malignancies generally occurwithin two years of cyclophosphamide treatment and occurs in patientswho previously had hemorrhagic cystitis (CYTOXAN (cyclophosphamide)package insert). Cyclophosphamide has toxic effects on the prostate andmale reproductive systems. Cyclophosphamide treatment can result in thedevelopment of sterility, and result in some degree of testicularatrophy.

Effects of KGF-2 Δ33 on Normal Bladder, Testes and Prostate ExperimentalDesign

Male Sprague-Dawley rats (160–220 g), (n=4 to 6/treatment group) wereused in these studies. KGF-2 Δ33 was administered at a dose of 5mg/kg/day. Daily ip or sc injections of recombinant KGF-2 Δ33 or buffer(40 mM sodium acetate+150 mM NaCl at pH 6.5) were administered for aperiod of 1–7 days and the rats were sacrificed the following day. Toexamine the reversibility of effects induced with KGF-2 Δ33, additionalanimals were injected ip daily for 7 days with KGF-2 Δ33 or buffer andsacrificed after a 7 day treatment-free period. On the day of sacrifice,rats were injected ip with 100 mg/kg of BrdU. Two hours later the ratswere overdosed with ether and selected organs removed. Samples oftissues were fixed in 10% neutral buffered formalin for 24 hours andparaffin embedded. To detect BrdU incorporation into replicating cells,five micron sections were subjected to immunohistochemical proceduresusing a mouse anti-BrdU monoclonal antibody and the ABC Elite detectionsystem. The sections were lightly counterstained with hematoxylin.

Sections were read by blinded observers. The number of proliferatingcells was counted in 10 random fields per animal at a 10× magnificationfor the prostate. To assess the effects of KGF-2 Δ33 in the bladder,cross-sections of these tissues were prepared and the number ofproliferating and non-proliferating cells were counted in ten randomfields at 20× magnification. The results are expressed as the percentageof labeled to unlabeled cells. Data are presented as mean±SEM.Statistical analyses (two-tailed unpaired t-test) were performed withthe StatView Software Package and statistical significance is defined asp<0.05.

Results

Bladder.

Intraperitoneal injection of KGF-2 Δ33 induced proliferation of bladderepithelial cells over the 7 day study period (solid squares, FIG. 52)but this did not influence the weight of the organ. Subcutaneousadministration elicited a small increase in proliferation but thisfailed to achieve statistical significance (solid circles, FIG. 52).

Prostate and Testes.

Both sc and ip administration of KGF-2 Δ33 induced significantproliferation of the prostate (FIG. 53) but this normalized after twoinjections. Prolonged ip treatment with KGF-2 Δ33 did not increase theweight of the prostate or testes.

Effects of KGF-2 Δ33 on Cyclophosphamide-Induced Hemorrhagic CystitisExperimental Design

Male Sprague Dawley rats (300–400g) (n=5/group) were injected i.v. viathe tail vein with buffer placebo or KGF-2 Δ33 at concentrations of 1 or5 mg/kg 24 hours prior to a 200 mg/kg i.p. injection ofcyclophosphamide. On the final day, 48 hours after cyclophosphamideinjection, rats were injected ip with 100 mg/kg of BrdU. Two hours laterthe rats were killed by CO₂ administration. Fixation of the bladder wasdone by direct injection of 10% formalin into the lumen of the bladderand rinsing of the exterior of the bladder with formalin. After 5minutes, the bladder and prostate were removed. The urinary bladder andprostate gland were paraffin embedded, cross-sectioned and stained withH&E and a mouse anti-BrdU monoclonal antibody. The extent of urothelialdamage was assessed using the following scoring system: Bladders weregraded by two independent observers to describe the extent of the lossof urothelium. (Urothelial damage was scored as 0, 25%, 50%, 75% and100% loss of the urothelium). In addition, the thickness of the bladderwall was measured at 10 random sites per section and expressed in μm.

Results

Macroscopic Observations

In rats treated with placebo and cyclophosphamide, bladders were thickand rigid. Upon injection of 10% formalin, very little expansion of thebladders was noted. However, in the groups pretreated with KGF-2 Δ33, agreater elasticity of the bladder was noted upon direct injection withformalin suggesting a lesser degree of fibrosis.

Microscopic Observations

FIG. 54 shows the results of KGF-2 Δ33 pretreatment on the extent ofulceration in the bladder. In normal rats treated with i.p. saline(saline control), the bladders appeared normal histologically and noulceration of the urothelium was observed. Administration of 200 mg/kgi.p. of cyclophosphamide resulted in ulceration of the bladderepithelium that was between 25 and 50% of the total epithelial area(with a mean of 37%). Administration of KGF-2 Δ33 24 hours prior tocyclophosphamide resulted in a significant reduction in the extent ofulceration (1 mg/kg 0.4% p=0.0128, and 5 mg/kg 5%, p=0.0338%) whencompared to placebo treated animals receiving cyclophosphamide.

FIG. 55 shows the effects of KGF-2 Δ33 on the thickness of the urinarybladder wall which includes epithelium, smooth muscle layers and theserosal surface. In groups treated with buffer alone, the thickness ofthe bladder wall is approximately 40 μm. Treatment with cyclophosphamideresults in a 5 fold increase in bladder wall thickness to 210 μm. KGF-2Δ33 pretreatment of cyclophosphamide treated animals resulted in asignificant inhibition of cyclophosphamide enlargement of the bladderwall (1 mg/kg 98.6 μm (p=0.007) and at 5 mg/kg 52.3 μm (p<0.0001)) whencompared to the cyclophosphamide treatment alone.

Microscopic Observations

Prostate Gland: In rats receiving buffer and cyclophosphamide, markedatrophy of the prostatic glands (acini) was observed accompanied byenlargement of interstitial spaces with remarkable edema when comparedto normals. In addition, epithelial cells lining the prostatic glandswere observed to be much shorter and less dense than in correspondingnormal prostatic tissue. KGF-2 Δ33 pretreatment at both 1 mg/kg and 5mg/kg displayed a normal histological appearance of the prostatic gland.No increase in the interstitial spaces or edema was observed, and theepithelial cells lining the prostatic glands were similar in size anddensity to normal prostatic tissue.

Conclusion

The results demonstrate that KGF-2 specifically induces proliferation ofbladder epithelial cells and the epithelial cells lining the prostaticglands. The results also demostrate that KGF-2 specifically results in asignificant reduction in the extent of ulceration incyclophosphamide-induced hemorrhagic cystitis.

EXAMPLE 31 Effect of KGF-2 on the Proliferation of Cells in Normal RatsIntroduction

KGF-2, a member of the FGF family, induces proliferation of normal humanand rat keratinocytes. It has approximately 57% homology to KGF-1 (amember of the FGF family). KGF-1 has been reported to induceproliferation of epithelia of many organs (Housley et al., Keratinocytegrowth factor induces proliferation of hepatocytes and epithelial cellsthroughout the rat gastrointestinal tract. J Clin Invest 94: 1764–1777(1994); Ulich et al., Keratinocyte growth factor is a growth factor fortype II pneumocytes in vivo. J Clin Invest 93: 1298–1306 (1994);Ulich etal., Keratinocyte growth factor is a growth factor for mammaryepithelium in vivo. The mammary epithelium of lactating rats isresistant to the proliferative action of keratinocyte growth factor. AmJ Pathol 144:862–868 (1994); Nguyen et al., Expression of keratinocytegrowth factor in embryonic liver of transgenic mice causes changes inepithelial growth and differentiation resulting in polycystic kidneysand other organ malformations. Oncogene 12:2109–2119 (1996); Yi et al.,Keratinocyte growth factor induces pancreatic ductal epithelialproliferation. Am J Pathol 145:80–85 (1994); and Yi et al., Keratinocytegrowth factor causes proliferation of urothelium in vivo. J Urology154:1566–1570 (1995)). We performed similar experiments with KGF-2 todetermine if it induces proliferation of normal epithelia in rats whenadministered systemically using sc and ip routes.

Methods:

Male Sprague-Dawley rats, weighing 160–220 g, were obtained from HarlanSprague Dawley for these studies. KGF-2 Δ33 (HG03411-E2) wasadministered at a dose of 5 mg/kg/day. Daily ip or sc injections ofKGF-2 Δ33 or recombinant buffer (40 mM sodium acetate+150 mM NaCl at pH6.5) were administered for a period of 1–7 days and the rats weresacrificed the following day (see below). To examine the reversibilityof effects induced with KGF-2 Δ33, additional animals were injected ipdaily for 7 days with KGF-2 Δ33 or buffer and sacrificed after a 7 daytreatment-free period.

On the day of sacrifice, rats were injected ip with 100 mg/kg of BrdU.Two hours later the rats were overdosed with ether and selected organsremoved. Samples of tissues were fixed in 10% neutral buffered formalinfor 24 hours and paraffin embedded. To detect BrdU incorporation intoreplicating cells, five micron sections were subjected toimmunohistochemical procedures using a mouse anti-BrdU monoclonalantibody (Boehringer Mannheim) and the ABC Elite detection system(Vector Laboratories). The sections were lightly counterstained withhematoxylin.

Sections were read by blinded observers. The number of proliferatingcells was counted in 10 random fields per animal at a 10× magnificationfor the following tissues: liver, pancreas, prostate, and heart. Tenrandom fields were used also for the lung analysis except theproliferation was quantitated at 20× magnification. Since the kidney hasmany functionally discrete areas, the proliferation was assessed in acoronal cross-section taken through the center of one kidney per animal.To assess the effects of KGF-2 Δ33 in the esophagus and bladder,cross-sections of these tissues were prepared and the number ofproliferating and non-proliferating cells were counted in ten randomfields at a 10× and 20× magnification, respectively. The results areexpressed as the percentage of labeled to unlabeled cells.

Data are presented as mean±SEM. Statistical analyses (two-tailedunpaired t-test) were performed with the StatView Software Package(Abacus Concepts, Inc., Berkeley, Calif.) and statistical significanceis defined as p<0.05.

Results

FIG. 56 shows an overview of the experimental protocol. Six animals wereused per group. However, during the analysis by the blinded observers itbecame clear that occasionally the BrdU injection was unsuccessful.Before the results were uncoded, the data from 8 rats out of 116 rats(or 7% of the animals) were excluded from the study and the resultantgroup sizes are shown in the Table below.

Group Sizes used in these Studies

n = Treatment Time ip sc KGF-2 Δ33 1 day 6 5 buffer 1 day 6 6 KGF-2 Δ332 days 6 4 buffer 2 days 6 6 KGF-2 Δ33 3 days 5 5 buffer 3 days 5 5KGF-2 Δ33 7 days 6 6 buffer 7 days 6 5 KGF-2 Δ33 7 days + 7 daystreatment-free 6 ND buffer 7 days + 7 days treatment-free 6 ND

Liver. When administered ip, KGF-2 Δ33 induced a rapid proliferation ofhepatocytes (solid squares) (FIG. 57) after 1 injection and thisaugmented mitotic activity persisted for three days, returning to normalafter 7 days of daily injections. In contrast to the dramatic effect ipadministration of KGF-2 exerted on the liver, when given sc (solidcircle, FIG. 57) this growth factor demonstrated minor effects.Proliferation was elevated after one day of treatment but returned tonormal values after two daily injections.

Pancreas. In contrast to the quickly reversible effects of ipadministered KGF-2 Δ33 on the liver, such injections inducedproliferation of the pancreas which continued over the 14 day studyperiod (solid squares, FIG. 58). Surprisingly, subcutaneousadministration of KGF-2 Δ33 (solid circles) failed to induceproliferation at any time point.

Kidney and Bladder. KGF-2 Δ33 induced proliferation of renal epitheliawhen given either by the sc or ip route but the former induced a greatereffect. SC administration induced a rapid increase in proliferation(solid circles) that peaked after 2 days which then returned to normalafter 7 daily treatments (FIG. 59). When KGF-2 Δ33 was given ip (solidsquares), there was a modest, but significant increase in proliferationseen at days 2 and 3 only. Intraperitoneal injection of KGF-2 Δ33 alsoinduced proliferation of bladder epithelial cells over the 7 day studyperiod (solid squares, FIG. 52). Subcutaneous administration elicited asmall increase in proliferation but this failed to achieve statisticalsignificance (solid circles, FIG. 52).

Prostate. Both sc and ip administration of KGF-2 Δ33 induced significantproliferation of the prostate (FIG. 53) but this normalized after twoinjections.

Esophagus. KGF-2 Δ33 given sc or ip elicited an early, short-livedincrease in the proliferation of the esophageal cells (1 and 2 days,respectively) that rapidly returned to normal (results not shown).

Other organs. Systemic administration of KGF-2 Δ33 by the ip and scroutes failed to elicit proliferation of the lung epithelia over a 7 daydosing period (results not shown).

2. Discussion

When administered in a sc route, we observed stimulation of normalepithelial proliferation in some organs (liver, kidney, esophagus, andprostate) but these effects, for the most part, were short-lived and allwere reversible. The proliferation in these organs reversed even duringdaily sc administration of KGF-2.

The route of administration had dramatic effects on the observedproliferation. While daily ip administration increased the rate of liverproliferation over a 3 day period, animals given KGF-2 sc dailyexhibited elevated rates after one day of treatment only. Even moresurprising was the response of the pancreas. When animals were givenKGF-2 ip, the pancreas exhibited a significantly elevated level ofproliferation over the 14 day study period. However, sc administrationof KGF-2 induced no increased mitotic activity in the pancreas.Likewise, ip, but not sc, treatment with KGF-2 elicited proliferation ofthe bladder mucosa.

IP administration of KGF-2 elicited a short-lived, small burst ofproliferation in the kidney that was centered in the region containingcollecting ducts. Daily sc treatment induced a prolonged, exaggeratedproliferation in this area.

EXAMPLE 32 Effects of KGF-2 Δ33 on Lung Cellular Proliferation FollowingIntratracheal Administration

The purpose of this example is to show that KGF-2 Δ33 is capable ofstimulating lung proliferation in normal rats following intratrachealadministration (administration of KGF-2 Δ33 directly to the lung).

Methods: Male Lewis rats (220–270 g), (n=5/treatment group) were used inthese studies. KGF-2 Δ33 or placebo (40 mM sodium acetate+150 mM NaCl atpH 6.5) was administered intratracheally at doses of 1 and 5 mg/kg in avolume of 0.6 mls followed by 3 mls of air. Treatments were administeredon day 1 and day 2 of the experimental protocol.

On day 3, the day of sacrifice, rats were injected ip with 100 mg/kg ofBrdU. Two hours later the rats were killed by CO₂ asphyxiation. Lungswere inflated with 10% buffered formalin via intratracheal catheter, andsaggital sections of lung were paraffin embedded. To detect BrdUincorporation into replicating cells, five micron sections weresubjected to immunohistochemical procedures using a mouse anti-BrdUmonoclonal antibody and the ABC Elite detection system. The sectionswere lightly counterstained with hematoxylin.

Sections were read by two blinded observers. The number of proliferatingcells was counted in 10 random fields per section at a 20×magnification. The results are expressed as the number of BrdU positivecells per field. Data are presented as mean±SEM. Statistical analyses(unpaired t-test) were performed with the Instat v2.0.1 and statisticalsignificance is defined as p<0.05.

Results: Intratracheal injection of KGF-2 Δ33 at 1 and 5 mg/kg resultedin an increase in proliferation of lung epithelial cells as shown inFIG. 60. KGF-2 Δ33 treatment resulted in statistically significantincreases in the number of BrdU positive cells/field at 1 mg/kg 23.4cells/field (p=0.0002) and at 5 mg/kg 10.3 cells/field (p=0.0003)relative to buffer controls of 1.58 cells per field.

EXAMPLE 33 Topical KGF-2 in Infected Incisional Wounds

Bacterial infection of wounds continues to be of great clinicalimportance. Under normal situations, the complex process of woundhealing progresses without difficulty. However, inoculation of a woundby bacteria causes an imbalance of cellular mediators in theinflammatory response resulting in delayed wound healing. Contaminationof the open wound inhibits the wound healing process as characterized bydecreased wound contraction, lower than normal wound collagen contentand decreased tensile strength. Male adult Sprague Dawley rats(n+10/group) were anesthetized with a combination of ketamine (53 mg/kgim) and xylazine (5.3 mg/kg im) on day 1. The dorsal region was shavedand disinfected with 70% alcohol. A full thickness (through theepidermis, dermis to the subcutaneous layer) 2.5 cm surgical wound wascreated starting approximately 1 cm below the shoulder blades using asterile no. 10 scalpel. Wounds were coated with 3 equidistant skinstaples. The incisions were then inoculated intraincisionally withStaphylococcus aureus (107 cfu/50 μl) in PBS. KGF-2 Δ33 was appliedtopically at the time of wounding (Day 0) at doses of 0.1, 1 and 10 μgper wound in a volume of 50 μl. Wounds were then covered with a gaspermeable occlusive dressing (Tegaderm). Animals were sacrificed on day5 by anesthesia with ketamine/xylazine followed by lethal intracardiacadministration of sodium pentobarbital (300 mg/kg). The middle 0.5 cmsegment of the wound was excised and snap frozen for collagendetermination. Two additional wound strips measuring 0.5 cm in widthwere excised. Excised wound strips were used for the study of breakingstrength using an Instron skin tensiometer. Breaking strength wasdefined as the greatest force withheld by each wound prior to ruptureusing and 11 1b load cell at a speed of 0 mm/sec. Two values for eachanimal were averaged to provide a mean breaking strength value perwound. Statistical analysis was done using an unpaired t test (mean±SE).

Intraincisional application of Staphylococcus aureus in the woundresulted in a significant impairment in wound healing as measured bybreaking strength (noninfected wound treated with bacteria vehicle136±6g; infected wound 87±6 g; p<0.0001 in one experiment; noninfectedwound treated with bacteria vehicle 200±14g; infected wound 154±10 gp=0.01 in another experiment). Topical administration of KGF-2 caused anincrease in breaking strength which was statistically significant at the0.1, 1 and 10 μg doses when compared with the KGF-2 buffer+S. aureuscontrol (KGF-2 0.1 μg 152±16 g (p=0.002); 1 μg 135±12 g (p=0.003); 10 μg158±10 g (p<0.0001) in one experiment; 0.1 μg 185±10g (p=0.03); 1 μg186±11g (p=0.03); 10 μg 190±7g p+0.009) in another experiment). Collagenanalysis of the middle 0.5 cm wound strip revealed that there wasincreased collagen content in KGF-2 treated wounds. However, whencompared with the buffer controls, a statistically significant increasein collagen content was not observed.

EXAMPLE 33 Proliferative Effect of Dosing i.v. Every Other Day With 1mg/kg of KGF-2 Δ33

Male Sprague Dawley rats were intravenously injected with either KGF-2Δ33 at a dose of 1 mg/kg, or buffer. The animals were injected eitherdaily or every other day. Each treatment group was injected for one weekand sacrificed at the end of the week. On the day of sacrifice, theanimals were injected i.p. with 100 mg/kg of BrdU. Two hours later, theanimals were sacrificed, and the serum was collected. Various tissueswere collected and fixed in 10% neutral buffered formalin. The tissueswere processed for histological evaluation. The tissues were stainedwith hematocylin and eosin, periodic-acid-Schiff, or alcian blue.Additional sections were subjected to immunohistochemical staining withan anti-BrdU antibody. Proliferation was quantitated using an imageanalysis spectrum, IPlab Spectrum. The serum chemistry analysis wasperformed using an automated chemistry analyzer. The followingparameters were quantitated: thyroid gland weight; proliferation ofgoblet cells in the small intestine (duodenum, jejunum and ileum);proliferation of goblet cells in the colon; proliferation in the parotidand submandibular glands; and serum chemistry analytes (glucose, BUN,calcium, total protein, albumin, alkaline phosphatase, alanineaminotransferase, aspartate aminotransferase, cholesterol, andtriglycerides).

In the small intestine and colon, daily treatment with KGF-2 caused asignificant increase in the number of goblet cells. The every other daytreatment did cause a slight increase in the number of goblet cells,however, it did not attain a statistically significant level. In thesalivary gland, an increase in cells was observed in the parotid glandonly. There was no difference between the treatment groups. There was anenlargement of the thyroid gland due to both dosing regimens. Themagnitude of this increase was greater in the daily treatment group.Daily treatment with KGF-2 resulted in statistically significantincrease in the following analytes: triglycerides, alkaline phosphatase,calcium, albumin, and total protein. The every other day treatment hadno effect on these analytes. Cholesterol levels were elevated in bothtreatment groups. However, the magnitude of the increase was greater inthe daily treatment group. Markers of cellular injury, such as ALT andAST, were similarly reduced in both treatment groups.

EXAMPLE 34 Formulating a Polypeptide

The KGF-2 composition will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the KGF-2 polypeptide alone), the site of delivery, themethod of administration, the scheduling of administration, and otherfactors known to practitioners. The “effective amount” for purposesherein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofKGF-2 administered parenterally per dose will be in the range of about 1μg/kg/day to 10 mg/kg/day of patient body weight, although, as notedabove, this will be subject to therapeutic discretion. More preferably,this dose is at least 0.01 mg/kg/day, and most preferably for humansbetween about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, KGF-2 is typically administered at a dose rate of about 1μg/kg/hour to about 50 μg/kg/hour, either by 1–4 injections per day orby continuous subcutaneous infusions, for example, using a mini-pump. Anintravenous bag solution may also be employed. The length of treatmentneeded to observe changes and the interval following treatment forresponses to occur appears to vary depending on the desired effect.

Pharmaceutical compositions containing KGF-2 are administered orally,rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), bucally, or as an oral or nasal spray.“Pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

KGF-2 is also suitably administered by sustained-release systems.Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547–556(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.Biomed. Mater. Res. 15:167–277 (1981), and R. Langer, Chem. Tech.12:98–105 (1982)), ethylene vinyl acetate (R. Langeret al.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomally entrapped KGF-2 polypeptides.Liposomes containing the KGF-2 are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688–3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030–4034 (1980); EP52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.Appl. 83–118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200–800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal secreted polypeptide therapy.

For parenteral administration, in one embodiment, KGF-2 is formulatedgenerally by mixing it at the desired degree of purity, in a unit dosageinjectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting KGF-2 uniformlyand intimately with liquid carriers or finely divided solid carriers orboth. Then, if necessary, the product is shaped into the desiredformulation. Preferably the carrier is a parenteral carrier, morepreferably a solution that is isotonic with the blood of the recipient.Examples of such carrier vehicles include water, saline, Ringer'ssolution, and dextrose solution. Non-aqueous vehicles such as fixed oilsand ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

KGF-2 is typically formulated in such vehicles at a concentration ofabout 0.1 mg/ml to 100 mg/ml, preferably 1–10 mg/ml, at a pH of about 3to 8. It will be understood that the use of certain of the foregoingexcipients, carriers, or stabilizers will result in the formation ofpolypeptide salts.

KGF-2 used for therapeutic administration can be sterile. Sterility isreadily accomplished by filtration through sterile filtration membranes(e.g., 0.2 micron membranes). Therapeutic polypeptide compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

KGF-2 polypeptides ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous KGF-2 polypeptide solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized KGF-2 polypeptide using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, KGF-2may be employed in conjunction with other therapeutic compounds.

The compositions of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the compositions of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the compositions of the invention are administered incombination with other members of the TNF family. TNF, TNF-related orTNF-like molecules that may be administered with the compositions of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), TR6(International Publication No. WO 98/30694), OPG, and neutrokine-alpha(International Publication No. WO 98/18921, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), and TR12, and soluble formsCD154, CD70, and CD153.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, tetracycline, metronidazole, amoxicillin,beta-lactamases, aminoglycosides, macrolides, quinolones,fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, andstreptomycin.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compostions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha.

In an additional embodiment, the compositions of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the compositions of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PIGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PIGF-2), as disclosed in Hauseret al., Gorwth Factors, 4:259–268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B-186 (VEGF-B186), as disclosed in InternationalPublication Number WO 96/26736; Vascular Endothelial Growth Factor-D(VEGF-D), as disclosed in International Publication Number WO 98/02543;Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/07832; and Vascular EndothelialGrowth Factor-E (VEGF-E), as disclosed in German Patent NumberDE19639601. The above mentioned references are incorporated herein byreference herein.

In an additional embodiment, the compositions of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

EXAMPLE 35 Method of Treating Decreased Levels of KGF-2

The present invention also relates to a method for treating anindividual in need of an increased level of KGF-2 activity in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of KGF-2 or an agonist thereof.

Moreover, it will be appreciated that conditions caused by a decrease inthe standard or normal expression level of KGF-2 in an individual can betreated by administering KGF-2, preferably in the secreted form. Thus,the invention also provides a method of treatment of an individual inneed of an increased level of KGF-2 polypeptide comprising administeringto such an individual a pharmaceutical composition comprising an amountof KGF-2 to increase the activity level of KGF-2 in such an individual.

For example, a patient with decreased levels of KGF-2 polypeptidereceives a daily dose 0.1–100 μg/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in the secreted form.The exact details of the dosing scheme, based on administration andformulation, are provided in Example 24.

EXAMPLE 36 Method of Treating Increased Levels of KGF-2

The present invention relates to a method for treating an individual inneed of a decreased level of KGF-2 activity in the body comprising,administering to such an individual a composition comprising atherapeutically effective amount of KGF-2 antagonist. Preferredantagonists for use in the present invention are KGF-2-specificantibodies.

Antisense technology is used to inhibit production of KGF-2. Thistechnology is one example of a method of decreasing levels of KGF-2polypeptide, preferably a secreted form, due to a variety of etiologies,such as cancer.

For example, a patient diagnosed with abnormally increased levels ofKGF-2 is administered intravenously antisense polynucleotides at 0.5,1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeatedafter a 7-day rest period if the treatment was well tolerated. Theformulation of the antisense polynucleotide is provided in Example 24.

EXAMPLE 37 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing KGF-2 polypeptides, onto a patient. Generally, fibroblastsare obtained from a subject by skin biopsy. The resulting tissue isplaced in tissue-culture medium and separated into small pieces. Smallchunks of the tissue are placed on a wet surface of a tissue cultureflask, approximately ten pieces are placed in each flask. The flask isturned upside down, closed tight and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted and thechunks of tissue remain fixed to the bottom of the flask and fresh media(e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) isadded. The flasks are then incubated at 37° C. for approximately oneweek.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219–25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding KGF-2 can be amplified using PCR primers whichcorrespond to the 5′ and 3′ end sequences respectively as set forth inExample 1. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector containsproperly inserted KGF-2.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the KGF-2 gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the KGF-2 gene(the packaging cellsare now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether KGF-2protein is produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

EXAMPLE 38 Gene Therapy Using Endogenous KGF-2 Gene

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous KGF-2 sequence with apromoter via homologous recombination as described, for example, in U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932–8935 (1989); and Zijlstra et al., Nature 342:435–438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not expressed in the cells, or is expressedat a lower level than desired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous KGF-2, flanking the promoter. The targeting sequence willbe sufficiently near the 5′ end of KGF-2 so the promoter will beoperably linked to the endogenous sequence upon homologousrecombination. The promoter and the targeting sequences can be amplifiedusing PCR. Preferably, the amplified promoter contains distinctrestriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ endof the first targeting sequence contains the same restriction enzymesite as the 5′ end of the amplified promoter and the 5′ end of thesecond targeting sequence contains the same restriction site as the 3′end of the amplified promoter. The amplified promoter and the amplifiedtargeting sequences are digested with the appropriate restrictionenzymes and subsequently treated with calf intestinal phosphatase. Thedigested promoter and digested targeting sequences are added together inthe presence of T4 DNA ligase. The resulting mixture is maintained underconditions appropriate for ligation of the two fragments. The constructis size fractionated on an agarose gel then purified by phenolextraction and ethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenousKGF-2 sequence. This results in the expression of KGF-2 in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the KGF-2 locus, plasmid pUC 18(MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMVpromoter is amplified by PCR with an XbaI site on the 5′ end and a BamHIsite on the 3′ end. Two KGF-2 non-coding sequences are amplified viaPCR: one KGF-2 non-coding sequence (KGF-2 fragment 1) is amplified witha HindIII site at the 5′ end and an Xba site at the 3′ end; the otherKGF-2 non-coding sequence (KGF-2 fragment 2) is amplified with a BamHIsite at the 5′ end and a HindIII site at the 3′ end. The CMV promoterand KGF-2 fragments are digested with the appropriate enzymes (CMVpromoter—XbaI and BamHI; KGF-2 fragment 1-XbaI; KGF-2 fragment 2-BamHI)and ligated together. The resulting ligation product is digested withHindIII, and ligated with the HindIII-digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250–300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14–20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37° C. The following day, the media is aspiratedand replaced with 10 ml of fresh media and incubated for a further 16–24hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

EXAMPLE 39 Method of Treatment Using Gene Therapy—In Vivo

Advances in gene research have resulted in the development of techniquesto deliver and express genes in human cells. The ideal goal for genetherapy is the delivery of normal genes in order to generate activeproteins and compensate for the lack of endogenous production (Gorecki,D. C. et al., Arch. Immunol. Ther. Exp. 45(5–6):375–381 (1997)).

Delivery of genes encoding cytokines or growth factors involved in thedifferent phases of wound healing and tissue repair have the potentialto modify the outcome of wound healing (Taub, P. J. et al., J. Reconst.Microsur. 14(6):387–390 (1998)). The use of cDNA of growth factors orother cytokines for wound healing and tissue repair has been extensivelydescribed (Tchorzewski, M. T. et al., J. Surg. Res. 77:99–103(1998)).Genes transferred by a vector can be used to generate new cell lines,identify transplanted cells and express growth factors or enzymes. Oneof the advantages of gene therapy is to achieve therapeuticconcentrations of gene-derived protein locally within the lesion site.Human recombinant KGF-2 protein has been shown to stimulate woundhealing of the skin, gastro-intestinal tract and other organ containingcells of epithelial origin. The use of KGF-2 gene is expected to havesimilar pharmacological profile as the recombinant protein. KGF-2 genemay be involved in events related to tissue repair such as cellproliferation, migration and the formation of extracellular matrix.

Transcribed and translated cDNA has been used to deliver genes to sitesof interest. Some examples of genes used in this fashion include aFGF,BMP-7 (Breitbart, A. S. et al., Ann. Plast. Surg. 24(5):488–495 (1999)).These cells have also been seeded into cell carriers includingbiodegradable matrices (ex. polyglycoloic acid), tissue substitutes orequivalents (ex. artificial skin), artificial organs, collagen-derivedmatrices, etc. Liposomes have been used to carry cDNA. PDGF-BB cDNA inhaemagglutinating virus of Japan (HVJ)-liposome suspension was studiedin the healing of patellar ligament (Nakamura et al., Gene Ther. 5(9):1165–1170 (1998)). Genes can also be delivered directly to the site ofaction by direct injection (ex. heart).

Thus, another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) KGF-2 sequences into an animal toincrease or decrease the expression of the KGF-2 polypeptide. The KGF-2polynucleotide may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the KGF-2 polypeptideby the target tissue. Such gene therapy and delivery techniques andmethods are known in the art, see, for example, WO90/11092, WO98/11779;U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Tabata, H., et al.,Cardiovasc. Res. 35(3):470–479 (1997), Chao, J., et al., Pharmacol. Res.35(6):517–522 (1997), Wolff, J. A., Neuromuscul. Disord. 7(5):314–318(1997), Schwartz B., et al., Gene Ther. 3(5):405–411 (1996), Tsurumi,Y., et al., Circulation 94(12):3281–3290 (1996) (incorporated herein byreference).

The KGF-2 polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The KGF-2 polynucleotideconstructs can be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the KGF-2 polynucleotides may also be delivered inliposome formulations (such as those taught in Felgner P. L. et al.,Ann. NY Acad. Sci. 772:126–139 (1995) and Abdallah B. et al., Biol. Cell85(1): 1–7 (1995)) which can be prepared by methods well known to thoseskilled in the art.

The KGF-2 polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapies techniques,one major advantage of introducing naked nucleic acid sequences intotarget cells is the transitory nature of the polynucleotide synthesis inthe cells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

The KGF-2 polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked KGF-2 polynucleotide injection, an effective dosage amountof DNA or RNA will be in the range of from about 0.05 g/kg body weightto about 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked KGF-2polynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected KGF-2 polynucleotide in muscle invivo is determined as follows. Suitable KGF-2 template DNA forproduction of mRNA coding for KGF-2 polypeptide is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The KGF-2 template DNA is injected in 0.1 ml of carrier in a1 cc syringe through a 27 gauge needle over one minute, approximately0.5 cm from the distal insertion site of the muscle into the knee andabout 0.2 cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for KGF-2 protein expression. A time course for KGF-2 proteinexpression may be done in a similar fashion except that quadriceps fromdifferent mice are harvested at different times. Persistence of KGF-2DNA in muscle following injection may be determined by Southern blotanalysis after preparing total cellular DNA and HIRT supernatants frominjected and control mice. The results of the above experimentation inmice can be use to extrapolate proper dosages and other treatmentparameters in humans and other animals using KGF-2 naked DNA.

EXAMPLE 40 KGF-2 Therapy for Inflammatory Bowel Disease

In this example, the inhibition of pathologic changes in colons of micecaused by exposure to dextran sodium sulfate (DSS) in drinking water bysystemic (intranasal) and intraperotineal administration of KGF-2polynucleotides is determined.

Intranasal administration. A polynucleotide encoding KGF-2 Δ33 isintroduced into the nasal passages of anaesthetized female Swiss Webstermice (n=10/group) through a blunted 26 gauge needle at a dosage of 1, 10or 100 μg of polynucleotide. Control polynucleotide is administered to aseparate group of mice. Five days after intranasal administration of thepolynucleotide, 5% DSS is added to the drinking water. Mice aremonitored for body weight, hematocrit, and stool score. After seven daysof exposure to DSS in the drinking water, mice are sacrificed.Histopathologic assessment of colon and small intestine is performed.RT-PCR analysis is performed to determine expression of KGF-2 in liver,spleen and colon.

Intraperotineal administration. A polynucleotide encoding KGF-2 Δ33 isinjected intraperitoneally into female Swiss Webster mice (n=10/group)through a blunted 26 gauge needle at a dosage of 1, 10 or 100 μg ofpolynucleotide on days 0 and 3. Control polynucleotide is administeredto a separate group of mice using and identical regimen. On day 7, 5%DSS is added to the drinking water. Mice are monitored for body weight,hematocrit, and stool score. On day 14, mice are sacrificed.Histopathologic assessment of colon and small intestine is performed.RT-PCR analysis is performed to determine expression of KGF-2 in liver,diaphragm and colon.

The studies described in this example test activity in KGF-2 Δ33polynucleotides. However, one skilled in the art could easily modify theexemplified studies to test the activity of other KGF-2 polynucleotides,including full length and mature KGF-2, KGF-2 Δ28, and polynucleotidesencoding amino acids 77 to 208, 80 to 208, and 93 to 208 of KGF-2; andKGF-2 polypeptides, variants, fragments, agonists, and/or antagonists;and any KGF-2 mutant described herein.

EXAMPLE 41 KGF-2 Therapy for Ocular Surface Disease

In this example, the effect of subconjuctival administration of A33KGF-2 polynucleotides on the conjunctiva, cornea or lacrimal gland ofrats is determined.

A polynucleotide encoding Δ33 KGF-2 is injected into the subconjuctivalspace of anaesthetized Female Sprague Dawley rats (150–200 g bodyweight, 6/treatment group) at a dosage of 1, 10 or 100 μg. Controlpolynucleotide is injected in a similar fashion to a separate group ofcontrol rats. Separate groups of rats are sacrificed at 3, 7 and 14 dayspost injection. BrdU is administered intraperitoneally to some of therats 30 minutes before euthanasia. The eye and surrounding tissues areremoved for histologic analysis. The extent of BrdU incorporation in theepithelial cells of the cornea, conjunctiva and lacrimal glands ismeasured. The thickness of the epithelial layer in the cornea andconjunctiva is assessed. The number of goblet cells in the conjunctivais measured.

The studies described in this example test activity in KGF-2 Δ33polynucleotides. However, one skilled in the art could easily modify theexemplified studies to test the activity of other KGF-2 polynucleotides,including full length and mature KGF-2, KGF-2 Δ28, and polynucleotidesencoding amino acids 77 to 208, 80 to 208, and 93 to 208 of KGF-2; andKGF-2 polypeptides, variants, fragments, agonists, and/or antagonists;as well as any KGF-2 mutant described herein.

EXAMPLE 42 KGF-2 Therapy for Salivary Gland Dysfunction

In this example, the effect of KGF-2 polynucleotide administration intothe papillae of the parotid salivary gland duct of normal rats on theepithelial cells of the ducts and acini of that gland is determined.

Female Sprague Dawley Rats (150–250 grams, 6/group) are anesthetized bythe intramuscular injection of ketamine and xylazine. A polynucleotideencoding Δ33 KGF-2 is introduced into the papilla of the parotidsalivary gland using a 30 gauge steel gavage needle, at a dosage of 1,10 or 100 μg. The polynucleotide is infused over a ten minute period ata rate of 1 μl per minute. Control polynucleotide is administered to aseparate group of rats. Separate groups of rats are sacrificed at 3, 7and 14 days after infusion. BrdU is administered intraperitoneally 30minutes before euthanasia. The salivary glands are weighed, and thenumber of BrdU-staining cells is counted on histologic section. In aseparate experiment, pilocarpine-stimulated saliva secretion is measuredin rats at 7 days after infusion.

The studies described in this example test activity in KGF-2 Δ33polynucleotides. However, one skilled in the art could easily modify theexemplified studies to test the activity of other KGF-2 polynucleotides,including full length and mature KGF-2, KGF-2 Δ28, and polynucleotidesencoding amino acids 77 to 208, 80 to 208, and 93 to 208 of KGF-2; andKGF-2 polypeptides, variants, fragments, agonists, and/or antagonists;as well as any KGF-2 mutant described herein.

EXAMPLE 43 KGF-2 Therapy for Dermal Wound Healing

In this example, the ability of KGF-2 polynucleotide to stimulate woundhealing in the normal rat and diabetic mice is determined.

Normal rat. Anesthetized female Sprague Dawley rats (175–250 μm6/treatment group) are wounded with 8 mm biopsy punches. Δ33 KGF-2polynucleotide (1, 10 or 30 μg) is delivered intradermally at 4different sites along the wound. Control polynucleotide is administeredin a similar manner to a separate group of rats. The wounds are coveredwith sterile ventilated fabric pads. After the pad is positioned,waterproof adhesive tape is wrapped around the midsection of the rat.Separate groups of rats are sacrificed at 2 and 5 days post wounding.The wound tissues are fixed in 10% formalin embedded in paraffin. BrdUincorporation in proliferating epithelial cells in pre-existing and newepidermis, and the length and thickness of the new epithelial tongue ismeasured.

Diabetic mice. Diabetic mice (db+/db+, 10/treatment group) andnondiabetic mice (db+/m+, 10/treatment group) are wounded with a 6 mmpunch wound in the dorsum. Δ33 KGF-2 polynucleotide (1, 10 or 30 μg) isdelivered intradermally at 4 different sites along the wound. Controlpolynucleotide is administered in a similar manner to a separate groupof mice. The wounds are covered with Tegaderm (diabetic mice) orTegaderm plus adhesive tape (nondiabetic mice). The wounds arephotographed on days 0, 3, 7, 10 and 14 post wounding. The surface areaof the wounds are measured by image analysis.

The studies described in this example test activity in KGF-2 Δ33polynucleotides. However, one skilled in the art could easily modify theexemplified studies to test the activity of other KGF-2 polynucleotides,including full length and mature KGF-2, KGF-2 Δ28, and polynucleotidesencoding amino acids 77 to 208, 80 to 208, and 93 to 208 of KGF-2; andKGF-2 polypeptides, variants, fragments, agonists, and/or antagonists;as well as any KGF-2 mutant described herein.

EXAMPLE 44 Constructs for KGF-2 Delivery

An appropriate construct for KGF-2 gene therapy delivery ispVGI.0-KGF-2. This construct contains the full native open reading frameof KGF-2 cloned into the expression vector pVGI.0. pVGI.0 contains akanamycin resistance gene, a CMV enhancer, and an RSV promoter.pVGI.0-KGF-2 was deposited at the American Type Culture CollectionPatent Depository, 10801 University Boulevard, Manassas, Va. 20110-2209,on Jun. 30, 1999, and given ATCC® Deposit No. PTA290. This construct wasmade by subcloning the KGF-2 ORF from a previously sequence verifiedKGF-2 construct into the expression vector pVGI-0, using methods wellknown in the art.

Another appropriate construct for KGF-2 delivery ispVGI-0-MPIFspKGF2Δ33. This construct contains the native sequence ofKGF-2 Δ33 fused to the MPIF (CK_(—)8) heterologous signal peptide clonedinto the expression vector pVGI-0. pVGI.0-MPIFspKGF2Δ33 was deposited atthe American Type Culture Collection Patent Depository, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, on Jun. 30, 1999, and given ATCC®Deposit No. PTA289. This construct was made using methods well known inthe art and the following primers:

5′ primer: GAGCGCGGATCCGCCACCATGAAGGTCTCCGTGGCTGCCCTCTCC (SEQ ID NO:149)TGCCTCATGCTTGTTACTGCCCTTGGATCTCAGGCCAGCTACAATCA CCTTCAAGGAGATG 3′primer: GAGCGC GGATCCCTATGAGTGTACCACCATTGGAAG (SEQ ID NO:150)

EXAMPLE 45 Angiogenesis During KGF-2 Gene Therapy

Characterization of the multiple aspects of microvascular physiology intransparent window systems in mice have provided valuable data onangiogenesis, inflammation, microvascular transport, tissue rejectionand tumor physiology. In this example, the development of vasculatureduring a wound healing response in implanted collagen gels is assessedthrough direct observation of the tissue and associated microvascularbed through an implanted skin window. This model is used to determine ifKGF-2 gene therapy can simultaneously induce an accelerated tissueregrowth and revascularization.

Skin biopsies from nude mice are digested in collagenase, the resultingcell suspensions washed and then cultured in DMEM with 10% FBS to obtaindermal fibroblasts. Confluent fibroblast cultures are transfected withKGF-2 or control polynucleotide then collected and washed in PBS. 106cells are suspended in 20 μl of collagen matrix. Samples of cellsuspension are removed for Western blot confirmation of KGF-2production. A 2 mm punch biopsy is made into an existing dorsal skinwindow and the skin sandwiched between two glass coverslips. The cellcollagen mixture is placed into the circular wound and the chambersealed. The implanted gels are observed at regular intervals for vesseldevelopment. Tissue regrowth into the wound is monitored as changes inthe optical density of the collagen gel over a three week period. Tissuefrom the dorsal chambers is removed following the conclusion of thestudy for histological evaluation. Control experiments involve theaddition of KGF-2 polypeptide or buffer into collagen gels in place offibroblasts.

Mouse preparation. The surgical procedures are performed in Swiss nudemice. For the surgical procedures, animals (20–30 g) are anesthetizedwith s.c. injection of a cocktail of 90 mg Ketamine and 9 mg Xylazineper kg body weight. All surgical procedures are performed under asepticconditions in a horizontal laminar flow hood, with all equipment beingsteam, gas or chemically sterilized. During surgery, the bodytemperature of the animals is kept constant by means of a heated worksurface. All mice are housed individually in miscroisolator cages andall manipulations are done in laminar flow hoods. Buprenorphine (0.1mg/kg q 12 h) is administered as an analgesic for 3 days postimplantation.

Mice are positioned such that the chamber is sandwiched between a doublelayer of skin that extends above the dorsal surface. One layer of skinis removed in a circular area ˜15 mm in diameter. The second layer(consisting of epidermis, fascia, and striated muscle) is positioned onthe frame of the chamber and covered with a sterile glass coverslip. Thechamber is held in place with nylon posts which pass through theextended skin and holes along the top of the chamber. After 3 days, thecoverslip is carefully removed and the gel inserted. A new, sterilecoverslip is then placed on the viewing surface. Measurements are madeby morphometric analysis using an Intensified CCD camera, S-VHSvideocassette recorder and direct digital image acquisition. Mice withimplanted changers were observed for 28 days.

Measurements. Mice are anesthetized with s.c. injection of a cocktail of90 mg Ketamine and 9 mg Xylazine per kg body weight, then positioned ona sterile plastic stage assembly. Vascular maps of the window are madeusing transillumination (dorsal skin window) or following an injectionof 1001 μl of BSA-FITC (1 mg/ml, i.v.) and epi-illumination. Videorecordings of vascular beds are made at a range of magnifications (from1× to 40×) as well as digital frames for off-line analysis. Angiogenesisdeterminations of implanted gels are made from offline analysis of videotapes.

The studies described in this example test activity in KGF-2 Δ33polynucleotides. However, one skilled in the art could easily modify theexemplified studies to test the activity of other KGF-2 polynucleotides,including full length and mature KGF-2, KGF-2 Δ28, and polynucleotidesencoding amino acids 77 to 208, 80 to 208, and 93 to 208 of KGF-2; andKGF-2 polypeptides, variants, fragments, agonists, and/or antagonists;as well as any of the KGF-2 mutants described herein.

EXAMPLE 46 KGF-2 Transgenic Animals

The KGF-2 polypeptides can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate transgenic animals. In a specific embodiment, techniquesdescribed herein or otherwise known in the art, are used to expresspolypeptides of the invention in humans, as part of a gene therapyprotocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691–698 (1994); Carver et al., Biotechnology(NY) 11:1263–1270 (1993); Wright et al., Biotechnology (NY)9:830–834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989));retrovirus mediated gene transfer into germ lines (Van der Putten etal., Proc. Natl. Acad. Sci., USA 82:6148–6152 (1985)), blastocysts orembryos; gene targeting in embryonic stem cells (Thompson et al., Cell56:313–321 (1989)); electroporation of cells or embryos (Lo, Mol. Cell.Biol. 3:1803–1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717–723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171–229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64–66 (1996); Wilmut et al., Nature 385:810–813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232–6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.

Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103–106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art. The contents of each of thedocuments recited in this paragraph is herein incorporated by referencein its entirety.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of KGF-2 polypeptides, studying conditions and/or disordersassociated with aberrant KGF-2 expression, and in screening forcompounds effective in ameliorating such conditions and/or disorders.

EXAMPLE 47 KGF-2 Knock-Out Animals

Endogenous KGF-2 gene expression can also be reduced by inactivating or“knocking out” the KGF-2 gene and/or its promoter using targetedhomologous recombination. (E.g., see Smithies et al., Nature 317:230–234(1985); Thomas & Capecchi, Cell 51:503–512 (1987); Thompson et al., Cell5:313–321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe KGF-2 polypeptides. The engineered cells which express andpreferably secrete the polypeptides of the invention can be introducedinto the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Knock-out animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of KGF-2 polypeptides, studying conditions and/or disordersassociated with aberrant KGF-2 expression, and in screening forcompounds effective in ameliorating such conditions and/or disorders.

EXAMPLE 48 Construction of KGF-2 Mutants

To create point mutants, the indicated primers were used in PCRreactions using standard conditions well known to those skilled in theart. The resulting products were restricted with either Nde and Asp718and cloned into pHE4; or with BamHI and Xba and cloned into pcDNA3; asindicated. Any of the described KGF-2 variants can be produced in othervectors, or by themselves, using methods well known in the art.

pHE4:KGF2:R80-S208 was constructed using following primers:

5′ primer: (SEQ ID NO:151) CCGGC CATATG CGTAAACTGTTCTCTTTCACC 3′ primer:(SEQ ID NO:152) CCGGC GGTACC TTATTATGAGTGTACCACCATTGG

pHE4:KGF2:A63-S208(R68G) was constructed using following primers:

5′ primer: (SEQ ID NO:153) GATCGC CATATG GCTGGTCGTCACGTTCGTTC 3′primer: (SEQ ID NO:154) GATCGC GGTACC TTATTATGAGTGTACCACCATTGGAAG

pHE4:KGF2:A63-S208(R68S) was constructed using following primers:

5′ primer: (SEQ ID NO:155) GATCGC CATATG GCTGGTCGTCACGTTCGTTC 3′primer: (SEQ ID NO:156) GATCGC GGTACC TTATTATGAGTGTACCACCATTGGAAG

pHE4:KGF2:A63-S208(R68A) was constructed using following primers:

5′ primer: (SEQ ID NO:157) GATCGC CATATG GCTGGTCGTCACGTTCGTTC 3′primer: (SEQ ID NO:158) GATCGC GGTACC TTATTATGAGTGTACCACCATTGGAAG

pHE4:KGF2:A63-S208(R78R80K81A) was constructed using following primers:

5′ primer: (SEQ ID NO:159) GATCGC CATATG GCTGGTCGTCACGTTCGTTC 3′primer: (SEQ ID NO:160) GATCGC GGTACC TTATTATGAGTGTACCACCATTGGAAG

pcDNA3:KGF2(K136137139144A) was constructed using following primers:

5′ primer: GATCGCGGATCCGCCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ IDNO:161) 3′ primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ IDNO:162)

pcDNA3:KGF2(K151153R155A) was constructed using following primers:

5′ primer: GATCGCGGATCCGCCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ IDNO:163) 3′ primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ IDNO:164)

pcDNA3:KGF2(R174K183A) was constructed using following primers:

5′ primer: GATCGCGGATCCGCCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ IDNO:165) 3′ primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ IDNO:166)

pcDNA3:KGF2(R187R188A) was constructed using following primers:

5′ primer: GATCGCGGATCCGCCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ IDNO:167) 3′ primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ IDNO:168)

pHE4:KGF2.A63(K136137139144A) was constructed using the followingprimers:

5′ primer: (SEQ ID NO:169) GATCGCCATATGGCTGGTCGTCACGTTCGTTC 3′primer: (SEQ ID NO:170) GATCGCGGTACCTTATTATGAGTGTACCACCATTGGAAG

pHE4:KGF2.A63(K151153R155A) was constructed using the following primers:

5′ primer: (SEQ ID NO:171) GATCGCCATATGGCTGGTCGTCACGTTCGTTC 3′primer: (SEQ ID NO:172) GATCGCGGTACCTTATTATGAGTGTACCACCATTGGAAG

EXAMPLE 49 Use of KGF-2 for Treating and/or Preventing Infertility

Implantation is the single most critical factor in a successfulpregnancy and is clinically and economically important. In humans, thegreatest fraction of the 70% loss in embryonic life occurs atimplantation. The mouse is the model of choice for studying mammalianimplantation. Three essential cell lineages differentiate and divide inthe peri-implantation mouse embryo: embryonic, placental and yolk sacprecursors. Fibroblast growth factor (FGF)-4 is essential fordevelopment of all three cell lineages.

It has been found, using a ‘transient transgenic’ approach to delivergain-of-function and loss-of-function (dominant negative) FGF receptorgenes, that endogenous FGF signaling is necessary for cell division ofall stem cells for the embryo and placenta lineages in the mouse embryostarting at the fifth cell division two days before implantation.

Interestingly, it has been found that null mutant for fgfr-2 and fgf4die in uteri within a day after implantation and the ICM dies. Beforethe embryo implants into the uterus cells in the embryonic lineage andin the placental lineage require FGF to continue proliferating.

It is possible that one or several of the other 19 FGF ligand isexpressed transiently in the mouse preimplantation embryo and thisligand delays the effect of the fgfr-2 and fgf4 null mutants until afterimplantation. We have tested for six FGF ligand using RT-PCR. To date,KGF-2 and FGF-8 are the only FGF ligands, besides FGF-4, detected in thepreimplantation embryo. KGF-2 mRNA is detected in the embryo after thetwo cell stage and through early post-implantation.

KGF-2 null mutants suggest that KGF-2 is not essential for survivalduring the expression of KGF-2 in peri-implantation mouse embryos (Minet al., 1998; Sekine et al., 1999). However, other FGF family membersmay compensate or be redundant for KGF-2 during peri-implantationembryonic development. Many redundant genetic effects have been observedduring analysis of null mutants in mice and compensation within a genefamily has also been observed (Thomas et al., 1995; Stein et al., 1994).KGF-2 may be more important in early development than is suggested bythe KGF-2 null mutants.

The best way to detect whether KGF-2 may have role in early developmentat a time when the null mutants suggest no essential function, is to dogain-of-function experiments. These experiments test whether KGF-2 hasan influence on growth of perimplantation embryos (Rappolee et al.,1994), on the placental/trophoblast cells in blastocyst outgrowths (Chaiet al., 1998) and in endoderm lineage cells in inner call mass (ICM)outgrowths (Rappolee et al., 1994). Loss-of-function tests can be donein a limited way by use of antisense oligonucleotides (Rappolee et al.,1992) or blocking antibodies (LaFleur et al., 1996). It is known thatthe embryos undergo size regulation, large positive and negative changesin cell number are homoeostatically regulated, soon after implantation(Rappolee, 1998). This suggests that small, sublethal KGF-2-dependenteffects might be totally missed in the KGF-2 null mutants. Loss- andgain-of-function experiments are use to test peri-implantation mouseembryos for the effects of KGF-2.

To date, the detection of mRNA for a growth factor in thepreimplantation mouse embryo has universally led to detection of thecorresponding protein. (Rappolee et al., 1998, 1992, 1994; reviewed inRappolee 1998, 1999). To determine whether KGF-2 protein is present (andwhere) in embryos where KGF-2 mRNA was detected, an antibody to KGF-2suitable for immunocytochemistry is used.

EXAMPLE 50 Detection of KGF-2 in a Clinical Sample

Purified Goat PAb is diluted to 2 μg/ml in the coating buffer (0.05 MNaHCO₃, Ph 9.5). 100 μl diluted antibody is added per well to an Immuno4 microplate. The microplate is stored overnight at 4° C. The antibodysolution is decanted from the plate. 200 μl of blocking buffer (1% drymilk (BioRad) in coating buffer) is added to each well. The plate isallowed to incubate at room temperature for 2 hours. The blocking bufferis decanted from the plate. The plate is vacuum aspirated and allowed todry completely in a vacuum chamber at 32° C. for 1.5 hours. The plate isremoved from the vacuum chamber and sealed in a mylar pouch with 3desiccant packs. The plate is stored at 4° C. until ready to be used.

KGF-2 is diluted to 16 ng/ml with diluent 1 (0.1% Tween 20, 1×PBS, 1%BSA, and 0.001% Thimerosal), then a subsequent 2.5× dilution is made forthe next 7 dilutions. The concentration range from 16 ng/ml to 0.026ng/ml is used as the standard. The background wells consist of diluentwithout protein.

The unknown samples are diluted 10×, 50×, and 250× with diluent 1. 100μl per well of the serial diluted standard solution and the unknownsamples are added to the coated ELISA plate. The plate is stored at 4°C. overnight.

The solutions are decanted from the plate. The plate is washed withwashing buffer (0.1% Tween 20 and 1×PBS) five times, using the WheatonInstrument set at 1.6 ml (each well receives 200 μl per wash). 15seconds of incubation of washing buffer is allowed between each wash.

The detector, biotinylated chicken anti-KGF-2 is diluted to 0.5 μg/ml indiluent 1. 100 μl of the diluted detector is added to each well. Theplate is incubated for 2 hours at room temperature. The solution isdecanted and the plate is washed with washing buffer 5 times, as before.15 seconds of incubation time is allowed between each wash.

Peroxidase streptavidin is diluted to 1:2000 in diluent 1. 100 μl perwell of the diluted peroxidase streptavidin is added to the plate andallowed to incubate at room temperature for 1 hour. The plate isdecanted and washed with washing buffer five times. 15 seconds ofincubation of washing buffer is allowed between each wash. The plate isnot allowed to dry.

Equal amounts of room temperature TMB peroxidase substrate and theperoxidase solution B (from the TMB Peroxidase Microwell SubstrateSystem, KPL) are mixed. 100 μl of the mixed solution is added to eachwell and the color is allowed to develop at room temperature for 10minutes. The color development is stopped by adding 50 μl of 1M H₂SO₄ toeach well. The plate is read at 450 nm.

EXAMPLE 51 Construction of E. coli Optimized Truncated KGF-2

In order to increase expression levels of a truncated KGF-2 in an E.coli expression system, the codons of the gene were optimized to highlyused E. coli codons.

For example, the following construct, termed pHE4:KGF-2.A63-S608, wasmade.

5′ CATATGGCTGGTCGTCACGTTCGTTCTTACA (SEQ ID NO: 173)ACCACCTGCAGGGTGACGTTCGTTGGCGTAAACT GTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGA ACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAAC AGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTG TAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATG GGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAA AACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAATAAGGTACC 3′

A plasmid comprising a cDNA having the nucleotide sequence of SEQ IDNO:173 was deposited as ATCC® Deposit No. PTA-2183 on Jul. 3, 2000, atthe American Type Culture Collection, Patent Depository, 10801University Boulevard, Manassas, Va. 20110-2209.

Another construct, termed pHE4:KGF-2.A63-S208 cod.opt, was constructedusing the following primers:

sense 5′ GACTACATATGGCTGGTCGTCACGTTCGTTC (SEQ ID NO: 174)TTACAACCACCTGCA GG3′ antisense 5′ CTAGTCTCTAGATTATTATGAGTGTACAACC (SEQID NO: 175) ATCGGCAGGAAGTGAG 3′

The nucleotide sequence of the pHE4:KGF-2.A63–208 cod.opt is as follows:

5′ ATGGCTGGTCGTCACGTTCGTTCTTACAACC (SEQ ID NO: 176)ACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTT CTCTTTCACCAAATACTTCCTGAAAATCGAAAAGAACGGTAAAGTTTCTGGTACCAAGAAAGAAAACT GCCCGTACTCTATCCTGGAAATCACCTCCGTTGAAATCGGTGTTGTAGCCGTTAAAGCCATCAACTCC AACTATTACCTGGCCATGAACAAAAAGGGTAAACTGTACGGCTCTAAAGAATTCAACAACGACTGCAA ACTGAAAGAACGTATCGAAGAGAACGGTTACAACACCTACGCATCCTTCAACTGGCAGCACAACGGTC GTCAGATGTACGTTGCACTGAACGGTAAAGGCGCTCCGCGTCGCGGTCAGAAAACCCGTCGCAAAAAC ACCTCTGCTCACTTCCTGCCGATGGTTGTACACTCATAATAA 3′

A plasmid comprising a cDNA having the nucleotide sequence of SEQ IDNO:176 was deposited as ATCC® Deposit No. PTA-2184 on Jul. 3, 2000, atthe American Type Culture Collection, Patent Depository, 10801University Boulevard, Manassas, Va. 20110-2209.

Both constructs described in this example are useful in the productionof KGF-2 polypeptides, for example, as described in Example 13.Nucleotides 4 to 444 of SEQ ID NO:173 and nucleotides 1 to 441 of SEQ IDNO:176 encode amino acids 63 to 208 of SEQ ID NO:2, plus an N-terminalmethionine.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

1. An isolated polynucleotide comprising a nucleic acid moleculeencoding a polypeptide comprising an first amino acid sequence at least95% identical to second amino acid sequence, wherein said second aminoacid sequence is selected from the group consisting of: (a) amino acidsAla (63) to Ser (208) of SEQ ID NO:2; (b) amino acids Ser (69) to Ser(208) of SEQ ID NO;2; (c) amino acids Ala (63) to Ser (208) of thepolypeptide encoded by the cDNA contained in ATCC Deposit No. 75977; and(d) amino acids Ser (69) to Ser (208) of the polypeptide encoded by thecDNA contained in ATCC Deposit No. 75977 wherein said polypeptidestimulates proliferation of epithelial cells.
 2. The isolatedpolynucleotide of claim 1, wherein said second amino acid sequence is(a).
 3. The isolated polynucleotide of claim 1, wherein said secondamino acid sequence is (b).
 4. The isolated polynucleotide of claim 1,wherein said second amino acid sequence is (c).
 5. The isolatedpolynucleotide of claim 1, wherein said second amino acid sequence is(d).
 6. The isolated polynucleotide of claim 1 further encoding a Metresidue at the N-terminus of said polypeptide.
 7. The isolatedpolynucleotide of claim 1 fused to a heterologous polynucleotide.
 8. Theisolated polynucleotide of claim 7 wherein said heterologouspolynucleotide encodes a heterologous polypeptide.
 9. A vectorcomprising the polynucleotide of claim
 1. 10. An isolated host cellcomprising the polynucleotide of claim 1 operably linked to a regulatorysequence.
 11. A method of producing a polypeptide comprising (a)culturing the host cell of claim 10 under conditions such that saidpolypeptide is expressed; and (b) recovering said polypeptide.
 12. Anisolated polynucleotide comprising a nucleic acid molecule encoding apolypeptide comprising an first amino acid sequence at least 97%identical to second amino acid sequence, wherein said second amino acidsequence is selected from the group consisting of: (a) amino acids Ala(63) to Ser (208) of SEQ ID NO:2; (b) amino acids Ser (69) to Ser (208)of SEQ ID NO:2; (c) amino acids Ala (63) to Ser (208) of the polypeptideencoded by the cDNA contained in ATCC Deposit No. 75977; and (d) aminoacids Ser (69) to Ser (208) of the polypeptide encoded by the cDNAcontained in ATCC Deposit No. 75977 wherein said polypeptide stimulatesproliferation of epithelial cells.
 13. The isolated polynucleotide ofclaim 12, wherein said second amino acid sequence is (a).
 14. Theisolated polynucleotide of claim 12, wherein said second amino acidsequence is (b).
 15. The isolated polynucleotide of claim 12, whereinsaid second amino acid sequence is (c).
 16. The isolated polynucleotideof claim 12, wherein said second amino acid sequence is (d).
 17. Theisolated polynucleotide of claim 12 further encoding a Met residue atthe N-terminus of said polypeptide.
 18. The isolated polynucleotide ofclaim 12 fused to a heterologous polynucleotide.
 19. The isolatedpolynucleotide of claim 18 wherein said heterologous polynucleotideencodes a heterologous polypeptide.
 20. A vector comprising thepolynucleotide of claim
 12. 21. An isolated host cell comprising thepolynucleotide of claim 12 operably linked to a regulatory sequence. 22.A method of producing a polypeptide comprising (a) culturing the hostcell of claim 21 under conditions such that said polypeptide isexpressed; and (b) recovering said polypeptide.